AmpFlSTR Identifiler Direct PCR Amplification Kit

Transcription

1 USER GUIDE AmpFlSTR Identifiler Direct PCR Amplification Kit for use with: 200 reaction kit (Part no ) 1000 reaction kit (Part no ) Publication Part Number Rev. J For Forensic or Paternity Use Only.

2 Information in this document is subject to change without notice. DISCLAIMER: LIFE TECHNOLOGIES CORPORATION AND/OR ITS AFFILIATE(S) DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED OR IMPLIED, INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE, OR NON- INFRINGEMENT. TO THE EXTENT ALLOWED BY LAW, IN NO EVENT SHALL LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) BE LIABLE, WHETHER IN CONTRACT, TORT, WARRANTY, OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING BUT NOT LIMITED TO THE USE THEREOF. Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept the terms and conditions of all applicable Limited Use Label Licenses. TRADEMARKS 2015 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. AmpliTaq Gold is a registered trademark of Roche Molecular Systems, Inc. Windows and Windows Vista are trademarks of Microsoft Corporation. FTA and Whatman are trademarks of Whatman International Ltd. Buccal DNA Collector is a trademark of Bode Technology Group, Inc. NUCLEIC-CARD and FLOQSwabs are trademarks of Copan Italia S.P.A.

3 Contents About This Guide Revision history Purpose CHAPTER 1 Overview Product overview Purpose Substrate examples Product description About the primers Loci amplified by the kit Allelic ladder Workflow Instrument and software overview Data collection and analysis software Instrument and software compatibility About multicomponent analysis How multicomponent analysis works Materials and equipment Kit contents and storage Standards for samples CHAPTER 2 Perform PCR Optimize PCR cycle number Select samples and prepare plates Determine optimum conditions Treated paper substrates: prepare reactions Sample prep guidelines Prepare the reactions Untreated paper substrates: prepare reactions Sample prep guidelines Prepare the reactions Swab substrates: prepare reactions Sample prep guidelines Prepare the sample lysate Prepare the reactions Store the sample lysate Perform PCR AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide 3

4 Contents CHAPTER 3 Perform Electrophoresis Allelic ladder requirements Section /3100-Avant and 3130/3130xl instruments Set up the 3100/3100-Avant and 3130/3130xl instruments for electrophoresis Reagents and parts Electrophoresis software setup and reference documents Prepare samples for electrophoresis on the 3100/3100-Avant or 3130/3130xl instruments Section /3500xL instruments Set up the 3500/3500xL instruments for electrophoresis Reagents and parts Electrophoresis software setup and reference documents Prepare samples for electrophoresis on the 3500/3500xL instruments Section instrument Set Up the 3730 instrument for electrophoresis Reagents and parts Electrophoresis software setup and reference documents Prepare samples for electrophoresis on the 3730 instrument CHAPTER 4 Analyze Data Section 4.1 GeneMapper ID Software Overview of GeneMapper ID Software Instruments Before you start Set up GeneMapper ID Software for data analysis File names Before using the software for the first time Import panels and bins Create an analysis method General tab settings Allele tab settings Peak Detector tab settings Peak Quality tab settings Quality Flags tab settings Create a size standard Analyze and edit sample files with GeneMapper ID Software Examine and edit a project For more information Section 4.2 GeneMapper ID-X Software Overview of GeneMapper ID-X Software Instruments Before you start AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide

5 Contents Set up GeneMapper ID-X Software for data analysis File names Before using the software for the first time Import panels, bins, and marker stutter Create an analysis method General tab settings Allele tab settings Peak Detector tab settings Peak Quality tab settings SQ & GQ tab settings Create a size standard Analyze and edit sample files with GeneMapper ID-X Software Examine and edit a project For more information CHAPTER 5 Experiments and Results Overview Importance of validation Experiment conditions Accuracy, precision, and reproducibility SWGDAM guideline SWGDAM guideline Accuracy Precision and size windows Extra peaks in the electropherogram Causes of extra peaks Stutter products Addition of 3 A nucleotide Artifacts Characterization of loci SWGDAM guideline Nature of the polymorphisms Mapping Species specificity SWGDAM Guideline Sensitivity SWGDAM guideline Blood on FTA cards Buccal cells on FTA or Indicating FTA cards and buccal cells on Bode DNA Collectors.. 81 Effect of DNA quantity on results Stability SWGDAM guideline DNA on FTA cards DNA on buccal swabs AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide 5

6 Contents Population data SWGDAM guideline Overview Population samples used in these studies Identifiler Direct Kit allele frequencies Evaluation of Hardy-Weinberg equilibrium Concordance studies Mutation rate Additional mutation studies Probability of identity Probability of paternity exclusion APPENDIX A Troubleshooting APPENDIX B Ordering Information Equipment and materials not included APPENDIX C Plate Layouts Example PCR plate layout Example electrophoresis plate layout APPENDIX D PCR Work Areas Work area setup and lab design PCR setup work area Amplified DNA work area APPENDIX E Safety Chemical safety Specific chemical handling Biological hazard safety Bibliography Documentation and Support Related documentation Obtain support Limited Product Warranty Index AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide

7 About This Guide IMPORTANT! Before using this product, read and understand the information the Safety appendix in this document. Revision history Revision Date Description A May 2009 New document. B August 2009 Add Experiments and Results chapter. C October 2009 Update screen shots for Panel Manager. D September 2010 Change copyright page information. E July 2011 Add 200-reaction kit, Bode Buccal DNA Collector, Prep-n-Go Buffer, and 3100-Avant, 3130, and 3500/3500xL Genetic Analyzer information. F October 2011 Add information for Prep-n-Go Buffer to Experiments and Results chapter. G March 2012 Change copyright page information. H May 2012 Add heat protocol for buccal swab lysate preparation. Add results for additional swab types. J February 2015 Add information for the ProFlex PCR System. Purpose The AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide provides information about our instruments, chemistries, and software associated with the AmpFlSTR Identifiler Direct PCR Amplification Kit. AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide 7

8 About This Guide Purpose 8 AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide

9 1 Overview Product overview Workflow Instrument and software overview Materials and equipment Product overview Purpose The AmpFlSTR Identifiler Direct PCR Amplification Kit is a short tandem repeat (STR) multiplex assay optimized to allow direct amplification of single-source: Blood and buccal samples on treated paper substrates without the need for sample purification. Blood and buccal samples collected on untreated paper substrates and treated with Applied Biosystems Prep-n-Go Buffer. Buccal samples collected on swab substrates and treated with Applied Biosystems Prep-n-Go Buffer The Identifiler Direct Kit amplifies 15 autosomal STR loci (D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vwa, TPOX, D18S51, D5S818, and FGA) and the sex-determining marker, Amelogenin, in a single PCR reaction. Substrate examples Product description Treated paper: Copan NUCLEIC-CARD system or Whatman FTA cards Untreated paper: Bode Buccal DNA Collector or 903 paper Swab: Copan 4N6FLOQSwabs The Identifiler Direct Kit contains all the necessary reagents for the amplification of human genomic DNA. The reagents are designed for use with the following Applied Biosystems instruments: Applied Biosystems 3100/3100-Avant Genetic Analyzer Applied Biosystems 3130/3130xl Genetic Analyzer Applied Biosystems 3500/3500xL Genetic Analyzer Applied Biosystems 3730 Genetic Analyzer GeneAmp PCR System 9700 with the Silver 96-Well Block GeneAmp PCR System 9700 with the Gold-plated Silver 96-Well Block Veriti 96-Well Thermal Cycler ProFlex PCR System AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide 9

10 1 Chapter 1 Overview Product overview About the primers Loci amplified by the kit The Identifiler Direct Kit employs the same primer sequences as used in the AmpFlSTR Identifiler PCR Amplification Kit. Degenerate primers for the loci D8S1179, vwa, and D16S539 are included in the AmpFlSTR Identifiler Direct Primer Set to address mutations in the primer binding sites. The addition of the degenerate primers allows for the amplification of those alleles in samples containing the mutations without altering the overall performance of the Identifiler Direct Kit. Non-nucleotide linkers are used in primer synthesis for the following loci: CSF1PO, D13S317, D16S539, D2S1338, and TPOX. For these primers, non-nucleotide linkers are placed between the primers and the fluorescent dye during oligonucleotide synthesis (Butler 2005, Grossman et al., 1994, and Baron et al., 1996). Non-nucleotide linkers enable reproducible positioning of the alleles to facilitate inter-locus spacing. The combination of a five-dye fluorescent system and the inclusion of non-nucleotide linkers allows for simultaneous amplification and efficient separation of the 15 STR loci and Amelogenin during automated DNA fragment analysis. Table 1 shows the loci amplified, their chromosomal locations, and the corresponding fluorescent marker dyes. The AmpFlSTR Identifiler Direct Allelic Ladder is used to genotype the analyzed samples. The alleles contained in the allelic ladder, and the genotype of the AmpFlSTR Identifiler Direct Control DNA 9947A, are also listed in the table. Table 1 AmpFlSTR Identifiler Direct PCR Amplification Kit loci and alleles Locus designation Chromosome location Alleles included in Allelic Ladder Dye label Control DNA 9947A D8S , 9 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 6-FAM 13, 13 D21S11 21q11.2-q21 24, 24.2, 25, 26, 27, 28, 28.2, 29, 29.2, 30, 30.2, 31, 30, , 32, 32.2, 33, 33.2, 34, 34.2, 35, 35.2, 36, 37, 38 D7S820 7q , 7, 8, 9, 10, 11, 12, 13, 14, 15 10, 11 CSF1PO 5q , 7, 8, 9, 10, 11, 12, 13, 14, 15 10, 12 D3S1358 3p 12, 13, 14, 15, 16, 17, 18, 19 VIC 14, 15 TH01 11p15.5 4, 5, 6, 7, 8, 9, 9.3, 10, 11, , 9.3 D13S317 13q , 9, 10, 11, 12, 13, 14, 15 11, 11 D16S539 16q24-qter 5, 8, 9, 10, 11, 12,13, 14, 15 11, 12 D2S1338 2q , 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 19, 23 D19S433 19q , 10, 11, 12, 12.2, 13, 13.2, 14, 14.2, 15, 15.2, 16, NED 14, , 17, 17.2 vwa 12p12-pter 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 17, 18 TPOX 2p23-2per 6, 7, 8, 9, 10, 11, 12, 13 8, 8 D18S51 18q21.3 7, 9, 10, 10.2, 11, 12, 13, 13.2, 14, 14.2, 15, 16, 17, 15, 19 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 Amelogenin X: p ; Y: p11.2 X, Y PET X D5S818 5q , 8, 9, 10, 11, 12, 13, 14, 15, 16 11, 11 FGA 4q28 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 26.2, 27, 28, 29, 30, 30.2, 31.2, 32.2, 33.2, 42.2, 43.2, 44.2, 45.2, 46.2, 47.2, 48.2, 50.2, , AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide

11 Chapter 1 Overview Product overview 1 Allelic ladder Figure 1 shows the allelic ladder for the Identifiler Direct Kit. See Allelic ladder requirements on page 28 for information on ensuring accurate genotyping. Figure 1 GeneMapper ID-X Software plot of the AmpFlSTR Identifiler Direct Allelic Ladder AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide 11

12 1 Chapter 1 Overview Workflow Workflow Perform PCR Obtain samples Treated or untreated paper substrates Obtain samples Swab substrates Prepare samples or Prepare samples Lyse in Prep-n-Go Buffer Harris Manual Punch CPA200 or CPA300 instrument Prepare reactions Untreated paper only: Prep-n-Go Buffer AmpFlSTR Identifiler Direct PCR Amplification Kit Prepare reactions AmpFlSTR Identifiler Direct PCR Amplification Kit Perform PCR GeneAmp PCR System 9700 Cycler ProFlex PCR System Veriti 96-Well Thermal Cycler Perform electrophoresis 3100/3100-Avant Genetic Analyzer 3130/3130xl Genetic Analyzer 3500/3500xL Genetic Analyzer 3730 Genetic Analyzer Analyze data GeneMapper ID-X Software GeneMapper ID Software 12 AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide

13 Chapter 1 Overview Instrument and software overview 1 Instrument and software overview This section provides information about the data collection and analysis software versions required to run the Identifiler Direct Kit on specific instruments. Data collection and analysis software Instrument and software compatibility The data collection software provides instructions to firmware running on the instrument and displays instrument status and raw data in real time. As the instrument measures sample fluorescence with its detection system, the data collection software collects the data and stores it. The data collection software stores information about each sample in a sample file (.fsa files for 31xx and 3730 instruments and.hid files for 3500 instruments), which is then analyzed by the analysis software. Instrument Data collection software Analysis software 3100/3100-Avant 1.1 (3100) 1.0 (3100-Avant) /3130xl /3500xL 3500 Series Data Collection Software v1.0 GeneMapper ID Software v3.2.1 GeneMapper ID-X Software v1.0.1 or later GeneMapper ID-X Software v1.2 or later We conducted validation studies for the Identifiler Direct Kit using these configurations. About multicomponent analysis Life Technologies fluorescent multi-color dye technology allows the analysis of multiple loci, including loci that have alleles with overlapping size ranges. Alleles for overlapping loci are distinguished by labeling locus-specific primers with different colored dyes. Multicomponent analysis is the process that separates the five different fluorescent dye colors into distinct spectral components. The four dyes used in the Identifiler Direct Kit to label samples are 6-FAM, VIC, NED, and PET dyes. The fifth dye, LIZ, is used to label the GeneScan 500 LIZ Size Standard or the GeneScan 600 LIZ Size Standard v2.0. How multicomponent analysis works Each of these fluorescent dyes emits its maximum fluorescence at a different wavelength. During data collection on Life Technologies instruments, the fluorescence signals are separated by a diffraction grating according to their wavelengths and projected onto a charge-coupled device (CCD) camera in a predictably spaced pattern. The 6-FAM dye emits at the shortest wavelength and is displayed as blue, followed by the VIC dye (green), NED dye (yellow), PET dye (red), and LIZ dye (orange). Although each of these dyes emits its maximum fluorescence at a different wavelength, there is some overlap in the emission spectra between the dyes (Figure 2). The goal of multicomponent analysis is to correct for spectral overlap. AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide 13

14 1 Chapter 1 Overview Materials and equipment Figure 2 Emission spectra of the five dyes used in the Identifiler Direct Kit Normalized Emission Dyes 6-FAM VIC NED PET LIZ Wavelength (nm) Materials and equipment Kit contents and storage The Identifiler Direct Kit contains sufficient quantities of the following reagents for 200 reactions (Part no ) or 1000 reactions (Part no ) at 25 µl/reaction. IMPORTANT! The fluorescent dyes attached to the primers are light-sensitive. Protect the primer set, amplified DNA, allelic ladder, and size standard from light when not in use. Keep freeze-thaw cycles to a minimum. Table 2 Kit Contents and Storage Component Description 200 reaction 1000 reaction Storage AmpFlSTR Identifiler Direct Master Mix AmpFlSTR Identifiler Direct Primer Set AmpFlSTR Identifiler Direct Control DNA 9947A AmpFlSTR Identifiler Direct Allelic Ladder Contains enzyme, salts, dntps, carrier protein, and 0.04% sodium azide Contains forward and reverse primers to amplify human DNA targets. Contains 2 ng/μl human female cell line DNA in 0.04% sodium azide and buffer. See Table 1 on page 10 for profile. Contains amplified alleles. See Table 1 on page 10 for a list of alleles included in the allelic ladder. 2 tubes, 1.25 ml each 2 tubes, 1.25 ml each 1 bottle, 12.5 ml 15 to 25 C upon receipt, 2 to 8 C after initial use 1 bottle, 12.5 ml 1 tube, 50.0 µl 1 tube, 50.0 µl 1 tube, 50.0 µl 1 tube, 100 µl The Control DNA 9947A is included at a concentration appropriate to its intended use as an amplification control (i.e., to provide confirmation of the capability of the kit reagents to generate a profile of expected genotype). The Control DNA 9947A is not designed to be used as a DNA quantitation control and laboratories may expect to see variation from the labelled concentration when quantitating aliquots of the Control DNA 9947A. 14 AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide

15 Chapter 1 Overview Materials and equipment 1 Standards for samples For the Identifiler Direct Kit, the panel of standards needed for PCR amplification, PCR product sizing, and genotyping are: AmpFlSTR Identifiler Direct Control DNA 9947A A positive control for evaluating the efficiency of the amplification step and STR genotyping using the AmpFlSTR Identifiler Direct Allelic Ladder. GeneScan 500 LIZ Size Standard or GeneScan 600 LIZ Size Standard v2.0 Used for obtaining sizing results. These standards, which have been evaluated as internal size standards, yield precise sizing results for Identifiler Direct Kit PCR products. Order the GeneScan 500 LIZ Size Standard (Part no ) or the GeneScan 600 LIZ Size Standard v2.0 (Part no ) separately. AmpFlSTR Identifiler Direct Allelic Ladder Developed for accurate characterization of the alleles amplified by the Identifiler Direct Kit. The Allelic Ladder contains most of the alleles reported for the 15 autosomal loci. Refer to page 10 for a list of the alleles included in the Allelic Ladder. AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide 15

16 1 Chapter 1 Overview Materials and equipment 16 AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide

17 2 Perform PCR Optimize PCR cycle number Treated paper substrates: prepare reactions Untreated paper substrates: prepare reactions Swab substrates: prepare reactions Perform PCR Optimize PCR cycle number Before using the Identifiler Direct Kit for the first time, perform a single initial sensitivity experiment to determine the appropriate cycle number to use during internal validation studies and operational use of the Identifiler Direct Kit. This experiment accounts for instrument-to-instrument and sample-to-sample variations. If you are processing multiple sample type and substrate combinations (for example, buccal samples on treated paper and buccal samples on swabs), perform separate sensitivity experiments for each sample type and substrate to be used for testing. The Identifiler Direct Kit is optimized to amplify unpurified: Single-source blood samples on treated paper or untreated paper Buccal samples on treated paper, untreated paper, or swabs When amplifying single-source, unpurified samples using the Identifiler Direct Kit, you should expect to see greater variation in peak height from sample to sample than is expected with purified samples. Careful optimization of the cycle number will help to minimize this variation. Select samples and prepare plates 1. Select 26 of each sample+substrate type. Ensure the selected samples represent a typical range of samples analyzed in your laboratory. IMPORTANT! The number of samples recommended for this study has been chosen to allow you to complete electrophoresis using a single 96-well plate, thus minimizing the impact of run-to-run variation on the results. Examples of PCR and electrophoresis plate layouts are provided on page Prepare the samples and the reactions as described in the protocols later in this chapter. Prepare sufficient PCR reagents to complete amplification of three replicate plates. 3. Create three identical PCR plates (see page 107 for a suggested plate layout). 4. Amplify each plate using a different cycle number to determine the optimum conditions for use in your laboratory. Suggested cycle numbers for different sample type and substrate combinations are listed below: AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide 17

18 2 Chapter 2 Perform PCR Optimize PCR cycle number Sample type Substrate Treated paper Untreated paper Swab Blood 25, 26, 27 cycles 25, 26, 27 cycles N/A Buccal 26, 27, 28 cycles 26, 27, 28 cycles 26, 27, 28 cycles Note: Our testing has not included blood samples on swab substrates. This sample type is not frequently used for the collection of database or casework reference samples. Note: To minimize the effect of instrument-to-instrument variation, use the same thermal cycler to amplify all three plates. To maximize result quality, prepare and amplify Plate 1 then repeat for Plates 2 and 3. Do not prepare all three plates simultaneously. Determine optimum conditions 1. Run the PCR products on the appropriate CE platform using the recommended protocol; see Chapter 3, Perform Electrophoresis on page Based on the results of the sensitivity study, select the appropriate PCR cycle number for future experiments. Our studies indicate the optimum PCR cycle number should generate profiles with the following heterozygote peak heights, with no instances of allelic dropout and minimal occurrence of off-scale allele peaks. Instrument Heterozygous peak height 31xx RFU 3500 Series ,000 RFU 18 AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide

19 Chapter 2 Perform PCR Treated paper substrates: prepare reactions 2 Treated paper substrates: prepare reactions Sample prep guidelines Prepare the reactions Do not add water to the wells on the reaction plate before adding the punches. If your laboratory is experiencing static issues with the paper discs, you may prepare and dispense the 25 µl reaction mix into the wells of the reaction plate before adding the punches. Make the punch as close as possible to the center of the sample to ensure optimum peak intensity. Increasing the size of the punch may cause inhibition during PCR amplification. For manual punching: Place the tip of a 1.2 mm Harris Micro-Punch on the card, hold the barrel of the Harris Micro-Punch (do not touch the plunger), gently press and twist 1/4-turn, then eject the punch in to the appropriate well on the reaction plate. For automated punching: Please refer to the User Guide of your automated or semi-automated disc punch instrument for proper guidance. 1. Add samples to the reaction plate: Well(s) Add the following to wells of a MicroAmp Optical 96-Well Reaction Plate... Negative control Test samples Positive control 1.2 mm blank disc 1.2 mm sample disc For 25 cycles 3 µl of Control DNA 9947A IMPORTANT! Do not add a blank disc to the positive control well. For 26 and 27 cycles For 28 cycles 2 µl of Control DNA 9947A 1 µl of Control DNA 9947A Note: The volumes of positive control are suggested amounts and may be adjusted if peak heights are too high or too low for your optimized cycle number. 2. Calculate the volume of each component needed to prepare the reactions, using the table below. Reaction component Volume per reaction Master Mix 12.5 µl Primer Set 12.5 µl Note: Include additional reactions in your calculations to provide excess volume for the loss that occurs during reagent transfers. IMPORTANT! The Identifiler Direct Kit has been optimized for a 25-µL PCR reaction volume to overcome the PCR inhibition expected when amplifying unpurified samples. Using a lower PCR reaction volume may reduce the ability of Identifiler Direct Kit chemistry to generate full STR profiles. AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide 19

20 2 Chapter 2 Perform PCR Treated paper substrates: prepare reactions 3. Prepare reagents. Thaw the Master Mix and the Primer Set, then vortex for 3 seconds and centrifuge briefly before opening the tubes or bottles. IMPORTANT! Thawing is required only during first use of the kit. After first use, reagents are stored at 2 to 8 C and, therefore, do not require subsequent thawing. Do not refreeze the reagents. 4. Pipet the required volumes of components into an appropriately sized polypropylene tube. 5. Vortex the reaction mix for 3 seconds, then centrifuge briefly. 6. Dispense 25 µl of the reaction mix into each reaction well of a MicroAmp Optical 96-Well Reaction Plate. 7. Seal the plate with MicroAmp Clear Adhesive Film or MicroAmp Optical Adhesive Film. IMPORTANT! If using the 9700 thermal cycler with silver or gold-plated silver block and adhesive clear film instead of caps to seal the plate wells, place a MicroAmp compression pad (Part no ) on top of the plate to prevent evaporation during thermal cycling. The Veriti Thermal Cycler does not require a compression pad. 8. Centrifuge the plate at 3000 rpm for about 20 seconds in a tabletop centrifuge with plate holders. 9. Amplify the samples in a GeneAmp PCR System 9700 with the silver or gold-plated silver 96-well block or a Veriti 96-well Thermal Cycler or a ProFlex PCR System as described in Perform PCR on page 26. IMPORTANT! The Identifiler Direct Kit is not validated for use with the GeneAmp PCR System 9700 with the aluminium 96-well block. Use of this thermal cycling platform may adversely affect performance of the Identifiler Direct Kit. 20 AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide

21 Chapter 2 Perform PCR Untreated paper substrates: prepare reactions 2 Untreated paper substrates: prepare reactions Sample prep guidelines Prepare the reactions Make the punch as close as possible to the center of the sample to ensure optimum peak intensity. Increasing the size of the punch may cause inhibition during PCR amplification. If you are using a Bode Buccal DNA Collector, make the punch as close as possible to the tip of the DNA collector to ensure optimum peak intensity. Increasing the size of the punch may cause inhibition during PCR amplification. For manual punching: Place the tip of a 1.2 mm Harris Micro-Punch on the card, hold the barrel of the Harris Micro-Punch (do not touch the plunger), gently press and twist 1/4-turn, then eject the punch in to the appropriate well on the reaction plate. Bode Buccal DNA Collector Take punch as close to the tip as possible For automated punching: Please refer to the User Guide of your automated or semi-automated disc punch instrument for proper guidance. 1. Add 2 µl of Prep-n-Go Buffer (Part no ) to the sample and negative control wells in a 96-well plate. Do not add Prep-n-Go Buffer to the positive control wells. 2. Add samples to the reaction plate: Well(s) Add the following to wells of a MicroAmp Optical 96-Well Reaction Plate... Negative control Test samples Positive control 1.2 mm blank disc 1.2 mm sample disc For 25 cycles 3 µl of Control DNA 9947A IMPORTANT! Do not add a blank disc to the positive control well. For 26 and 27 cycles For 28 cycles 2 µl of Control DNA 9947A 1 µl of Control DNA 9947A Note: The volumes of positive control are suggested amounts and may be adjusted if peak heights are too high or too low for your optimized cycle number. 3. Centrifuge the plate to ensure the punches are immersed in the Prep-n-Go Buffer. 4. Calculate the volume of each component needed to prepare the reactions, using the table below. Reaction component Volume per reaction Master Mix 12.5 µl Primer Set 12.5 µl Note: Include additional reactions in your calculations to provide excess volume for the loss that occurs during reagent transfers. AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide 21

22 2 Chapter 2 Perform PCR Untreated paper substrates: prepare reactions IMPORTANT! The Identifiler Direct Kit has been optimized for a 25-µL PCR reaction volume to overcome the PCR inhibition expected when amplifying unpurified samples. Using a lower PCR reaction volume may reduce the ability of Identifiler Direct Kit chemistry to generate full STR profiles. 5. Prepare reagents. Thaw the Master Mix and the Primer Set, then vortex for 3 seconds and centrifuge briefly before opening the tubes or bottles. IMPORTANT! Thawing is required only during first use of the kit. After first use, reagents are stored at 2 to 8 C and, therefore, do not require subsequent thawing. Do not refreeze the reagents. 6. Pipet the required volumes of components into an appropriately sized polypropylene tube. 7. Vortex the reaction mix for 3 seconds, then centrifuge briefly. 8. Dispense 25 µl of the reaction mix into each reaction well of a MicroAmp Optical 96-Well Reaction Plate. 9. Seal the plate with MicroAmp Clear Adhesive Film or MicroAmp Optical Adhesive Film. IMPORTANT! If using the 9700 thermal cycler with silver or gold-plated silver block and adhesive clear film instead of caps to seal the plate wells, place a MicroAmp compression pad (Part no ) on top of the plate to prevent evaporation during thermal cycling. The Veriti Thermal Cycler does not require a compression pad. 10. Centrifuge the plate at 3000 rpm for about 20 seconds in a tabletop centrifuge with plate holders. 11. Amplify the samples in a GeneAmp PCR System 9700 with the silver or gold-plated silver 96-well block or a Veriti 96-well Thermal Cycler or a ProFlex PCR System as described in Perform PCR on page 26. IMPORTANT! The Identifiler Direct Kit is not validated for use with the GeneAmp PCR System 9700 with the aluminium 96-well block. Use of this thermal cycling platform may adversely affect performance of the AmpFlSTR Identifiler Direct PCR Amplification Kit. 22 AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide

23 Chapter 2 Perform PCR Swab substrates: prepare reactions 2 Swab substrates: prepare reactions Note: Performance verification and optimization experiments for this protocol were conducted using Copan 4N6FLOQSwabs, OmniSwabs, and Puritan swabs air-dried and stored at room temperature for up to three months. Sample prep guidelines Detach buccal swab heads from the swab shaft for lysis. Lysis is performed under heated conditions using Prep-n-Go Buffer (Part no for buccal swabs) in either of the following formats: 1.5 ml tubes with a heat block (VWR Scientific Select dry heat b lock or similar) 96-well deep-well plate (Part no ) with an oven and a metal plate adaptor (Robbins Scientific Model 400 Hybridization Incubator or similar, Agilent Benchtop Rack for 200 µl Tubes/V Bottom Plates (metal) Part no or similar) IMPORTANT! Do not use a plastic plate adaptor. For optimum performance, lysis of a whole swab is recommended. To preserve the sample, evaluate lysis of a half swab. Prepare the sample lysate 1. Preheat the heat block to 90 C or the oven with metal plate adaptor to 99 C. 2. Add 400 µl Prep-n-Go Buffer (for buccal swabs, Part. no ) to 1.5 ml tubes or the appropriate wells of a 96-well deep-well plate (Part no ). 3. Into each tube or well, put the entire head of each swab. If you are using tubes, cap the tubes. Let the tubes or plate stand for 20 minutes in the preheated heat block or oven to lyse the sample. 4. After 20 minutes, remove the tubes or the deep-well plate from the heat block or oven. Note: To minimize the risk of contamination, do not remove the swab heads from the sample lysate plate before transferring the lysate. 5. Let the lysate stand at room temperature for at least 15 minutes to cool the lysate (for accurate pipetting). 6. Transfer the sample lysate out of the sample plate into tubes or plates for storage, then discard the deep-well plate containing the swab heads. 7. Proceed to the next section to prepare the reactions or see Store the sample lysate on page 25. Prepare the reactions 1. Calculate the volume of each component needed to prepare the reactions, using the table below. Reaction component Volume per reaction Master Mix 12.5 µl Primer Set 12.5 µl AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide 23

24 2 Chapter 2 Perform PCR Swab substrates: prepare reactions Note: Include additional reactions in your calculations to provide excess volume for the loss that occurs during reagent transfers. IMPORTANT! This kit has been optimized for a 25-µL PCR reaction volume to overcome the PCR inhibition expected when amplifying unpurified samples. Using a lower PCR reaction volume may reduce the ability of Identifiler Direct Kit chemistry to generate full STR profiles. 2. Prepare reagents. Thaw the Master Mix and the Primer Set, then vortex for 3 seconds and centrifuge briefly before opening the tubes or bottles. IMPORTANT! Thawing is required only during first use of the kit. After first use, reagents are stored at 2 to 8 C and, therefore, do not require subsequent thawing. Do not refreeze the reagents. 3. Pipet the required volumes of components into an appropriately sized polypropylene tube. 4. Vortex the reaction mix for 3 seconds, then centrifuge briefly. 5. Dispense 25 µl of the reaction mix into each reaction well of a MicroAmp Optical 96-Well Reaction Plate. 6. Add samples to the reaction plate: Well(s) Add the following to wells of a MicroAmp Optical 96-Well Reaction Plate... Negative control 3 µl of Prep-n-Go Buffer Test samples 3 µl of lysate Positive control For 25 cycles 3 µl of Control DNA 9947A For 26 and 27 cycles 2 µl of Control DNA 9947A For 28 cycles 1 µl of Control DNA 9947A Note: The volumes of positive control are suggested amounts and may be adjusted if peak heights are too high or too low for your optimized cycle number. 7. Seal the plate with MicroAmp Clear Adhesive Film or MicroAmp Optical Adhesive Film. IMPORTANT! If using the 9700 thermal cycler with silver or gold-plated silver block and adhesive clear film instead of caps to seal the plate wells, place a MicroAmp compression pad (Part no ) on top of the plate to prevent evaporation during thermal cycling. The Veriti Thermal Cycler does not require a compression pad. 8. Vortex the reaction mix at medium speed for 3 seconds. 9. Centrifuge the plate at 3000 rpm for about 20 seconds in a tabletop centrifuge with plate holders. 24 AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide

25 Chapter 2 Perform PCR Swab substrates: prepare reactions Amplify the samples in a GeneAmp PCR System 9700 with the silver or gold-plated silver 96-well block or a Veriti 96-well Thermal Cycler or a ProFlex PCR System as described in Perform PCR on page 26. IMPORTANT! The Identifiler Direct Kit is not validated for use with the GeneAmp PCR System 9700 with the aluminium 96-well block. Use of this thermal cycling platform may adversely affect performance of the Identifiler Direct Kit. Store the sample lysate Cap the sample lysate storage tubes or seal the sample lysate storage plate with MicroAmp Clear Adhesive Film. Store the sample lysate as needed: If you are storing the sample lysate... Then place at... <2 weeks 2 to 8 C >2 weeks 15 to 25 C These storage recommendations are preliminary pending the results of ongoing stability studies. The effects of multiple freeze-thaw cycles on the lysate have not been fully evaluated. Therefore, multiple freeze-thaw cycles are not recommended. AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide 25

26 2 Chapter 2 Perform PCR Perform PCR Perform PCR 1. Program the thermal cycling conditions. When using the GeneAmp PCR System 9700 with either 96-well silver or gold-plated silver block, select the 9600 Emulation Mode. When using the Veriti 96-Well Thermal Cycler, refer to the following document for instructions on how to configure the Veriti instrument to run in the 9600 Emulation Mode: User Bulletin: Veriti 96-Well Thermal Cycler AmpFlSTR Kit Validation (PN ). When using the ProFlex PCR System, refer to the ProFlex PCR System Kit Validation User Bulletin (Pub. no ) for more information. Initial incubation step Optimum cycle number Denature Anneal Extend Final extension Final hold HOLD CYCLE HOLD HOLD 95 C 11 min 94 C 20 sec 59 C 2 min 72 C 1 min 60 C 25 min 4 C Determine the optimum cycle number for your laboratory according to the instructions on page Load the plate into the thermal cycler and close the heated cover. 3. Start the run. 4. On completion of the run, store the amplified DNA. If you are storing the DNA... Then place at... <2 weeks 2 to 8 C >2 weeks 15 to 25 C IMPORTANT! Protect the amplified products from light. 26 AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide

27 3 Perform Electrophoresis Allelic ladder requirements Section /3100-Avant and 3130/3130xl instruments Set up the 3100/3100-Avant and 3130/3130xl instruments for electrophoresis...29 Prepare samples for electrophoresis on the 3100/3100-Avant or 3130/3130xl instruments Section /3500xL instruments Set up the 3500/3500xL instruments for electrophoresis Prepare samples for electrophoresis on the 3500/3500xL instruments Section instrument Set Up the 3730 instrument for electrophoresis Prepare samples for electrophoresis on the 3730 instrument AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide 27

28 3 Chapter 3 Perform Electrophoresis Allelic ladder requirements Allelic ladder requirements To accurately genotype samples, you must run an allelic ladder sample along with the unknown samples. Instrument Number of allelic ladders to run One injection equals Number of samples per allelic ladder(s) 3100-Avant or per 4 injections 4 samples 15 samples + 1 allelic ladder 3100 or 3130xl 1 per injection 16 samples 15 samples + 1 allelic ladder per 3 injections 8 samples 23 samples + 1 allelic ladder 3500xL 1 per injection 24 samples 23 samples + 1 allelic ladder per injection 48 samples 46 samples + 2 allelic ladders IMPORTANT! Variation in laboratory temperature can cause changes in fragment migration speed and sizing variation between both single- and multiple-capillary runs (with larger size variations seen between samples injected in multiple-capillary runs). We recommend the above frequency of allelic ladder injections, which should account for normal variation in run speed. However, during internal validation studies, verify the required allelic ladder injection frequency to ensure accurate genotyping of all samples in your laboratory environment. It is critical to genotype using an allelic ladder run under the same conditions as the samples, because size values obtained for the same sample can differ between instrument platforms because of different polymer matrices and electrophoretic conditions. 28 AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide

29 Section /3100-Avant and 3130/3130xl instruments Set up the 3100/3100-Avant and 3130/3130xl instruments for electrophoresis 3 Section /3100-Avant and 3130/3130xl instruments Set up the 3100/3100-Avant and 3130/3130xl instruments for electrophoresis Reagents and parts Appendix B, Ordering Information on page 103 lists the required materials not supplied with the Identifiler Direct Kit. IMPORTANT! The fluorescent dyes attached to the primers are light-sensitive. Protect the primer set, amplified DNA, allelic ladder, and size standard from light when not in use. Keep freeze-thaw cycles to a minimum and 3130 Instruments Electrophoresis software setup and reference documents The following table lists data collection software and the run modules that can be used to analyze Identifiler Direct Kit PCR products. For details on the procedures, refer to the documents listed in the table. Genetic Analyzer Data Collection Software Operating System Run modules and conditions References Applied Biosystems 3100-Avant 1.0 Windows NT GeneScan36Avb_DyeSetG5Module Injection condition: 3 kv/5sec GS600v2.0Analysis.gsp 3100/3100-Avant Genetic Analyzers Protocols for Processing AmpFlSTR PCR Amplification Kit PCR Products User Bulletin (Part no ) Applied Biosystems Windows 2000 HIDFragmentAnalysis36_POP4_1 Injection condition: 3kV/10 sec Dye Set G5 3100/3100-Avant Genetic Analyzers Using Data Collection Software v2.0, Protocols for Processing AmpFlSTR PCR Amplification Kit PCR Products User Bulletin (Part no ) 1.1 Windows NT GeneScan36vb_DyeSetG5Module Injection condition: 3kV/10 sec GS600v2.0Analysis.gsp 3100/3100-Avant Genetic Analyzers Protocols for Processing AmpFlSTR PCR Amplification Kit PCR Products User Bulletin (Part no ) Applied Biosystems 3130/3130xl 3.0 Windows XP HIDFragmentAnalysis36_POP4_1 Injection conditions: 3130 = 3 kv/5 sec 3130xl = 3 kv/10 sec Dye Set G5 Applied Biosystems 3130/3130xl Genetic Analyzers Using Data Collection Software v3.0, Protocols for Processing AmpFlSTR PCR Amplification Kit PCR Products User Bulletin (Part no ) We conducted validation studies for the Identifiler Direct Kit using this configuration. AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide 29

30 3 Chapter 3 Perform Electrophoresis Prepare samples for electrophoresis on the 3100/3100-Avant or 3130/3130xl instruments Prepare samples for electrophoresis on the 3100/3100-Avant or 3130/3130xl instruments Prepare the samples for electrophoresis immediately before loading. 1. Calculate the volume of Hi-Di Formamide and size standard needed to prepare the samples: Reagent Volume per reaction Reagent Volume per reaction GeneScan 500 LIZ Size Standard 0.3 µl OR GeneScan 600 LIZ Size Standard v µl Hi-Di Formamide 8.7 µl Hi-Di Formamide 8.5 µl Note: Include additional samples in your calculations to provide excess volume for the loss that occurs during reagent transfers. IMPORTANT! The volume of size standard indicated in the table is a suggested amount. Determine the appropriate amount of size standard based on your experiments and results. 2. Pipet the required volumes of components into an appropriately sized polypropylene tube. 3. Vortex the tube, then centrifuge briefly. 4. Into each well of a MicroAmp Optical 96-Well Reaction Plate, add: 9 µl of the formamide:size standard mixture 1 µl of PCR product or Allelic Ladder Note: For blank wells, add 10 µl of Hi-Di Formamide. 5. Seal the reaction plate with appropriate septa, then briefly vortex and centrifuge the plate to ensure that the contents of each well are mixed and collected at the bottom. 6. Heat the reaction plate in a thermal cycler for 3 minutes at 95 C. 7. Immediately place the plate on ice for 3 minutes. 8. Prepare the plate assembly on the autosampler. 9. Start the electrophoresis run. 30 AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide

31 Section /3500xL instruments Set up the 3500/3500xL instruments for electrophoresis 3 Section /3500xL instruments Set up the 3500/3500xL instruments for electrophoresis Reagents and parts Electrophoresis software setup and reference documents Appendix B, Ordering Information on page 103 lists the required materials not supplied with the Identifiler Direct Kit. IMPORTANT! The fluorescent dyes attached to the primers are light-sensitive. Protect the primer set, amplified DNA, allelic ladder, and size standard from light when not in use. Keep freeze-thaw cycles to a minimum. The following table lists data collection software and the run modules that you can use to analyze Identifiler Direct Kit PCR products. For details on the procedures, refer to the documents listed in the table. 3500/3500 xl Instruments Genetic Analyzer Data Collection Software Operating System Run modules and conditions References Applied Biosystems 3500 Applied Biosystems 3500xL 3500 Data Collection Software v1.0 Windows XP or Windows Vista HID36_POP4 Injection conditions: 1.2kV/15 sec Dye Set G5 HID36_POP4 Injection conditions: 1.2kV/24 sec Dye Set G5 Applied Biosystems 3500/ 3500xL Genetic Analyzer User Guide (Part no ) 3500 and 3500xL Genetic Analyzers Quick Reference Card (Part no ) Prepare samples for electrophoresis on the 3500/3500xL instruments Prepare the samples for electrophoresis immediately before loading. 1. Calculate the volume of Hi-Di Formamide and GeneScan 600 LIZ Size Standard v2.0 needed to prepare the samples: Reagent Volume per reaction GeneScan 600 LIZ Size Standard v µl Hi-Di Formamide 8.5 µl Note: Include additional samples in your calculations to provide excess volume for the loss that occurs during reagent transfers. IMPORTANT! The volume of size standard indicated in the table is a suggested amount. Determine the appropriate amount of size standard based on your experiments and results. AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide 31

32 3 Chapter /3500xL Instruments Prepare samples for electrophoresis on the 3500/3500xL instruments 2. Pipet the required volumes of components into an appropriately sized polypropylene tube. 3. Vortex the tube, then centrifuge briefly. 4. Into each well of a MicroAmp Optical 96-Well Reaction Plate, add: 9 µl of the formamide:size standard mixture 1 µl of PCR product or Allelic Ladder Note: For blank wells, add 10 µl of Hi-Di Formamide. 5. Seal the reaction plate with appropriate septa, then briefly vortex and centrifuge the plate to ensure that the contents of each well are mixed and collected at the bottom. 6. Heat the plate in a thermal cycler for 3 minutes at 95 C. 7. Immediately place the plate on ice for 3 minutes. 8. Place the sample tray on the autosampler. 9. Start the electrophoresis run. 32 AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide

33 Section instrument Set Up the 3730 instrument for electrophoresis 3 Section instrument Set Up the 3730 instrument for electrophoresis Reagents and parts Appendix B, Ordering Information on page 103 lists the required materials not supplied with the Identifiler Direct Kit. Electrophoresis software setup and reference documents Operating system Data collection software IMPORTANT! The fluorescent dyes attached to the primers are light-sensitive. Protect the primer set, amplified DNA, allelic ladder, and size standard from light when not in use. Keep freeze-thaw cycles to a minimum. The following table lists data collection software and the run modules that you can use to analyze Identifiler Direct Kit PCR products. For details on the procedures, refer to the documents listed in the table. Run module References 3730 Instruments Windows XP 3.0 GeneMapper_36_POP7 Dye Set G5_RCT Applied Biosystems 3730 DNA Analyzer Human Identification Validation Report We conducted concordance studies for the Identifiler Direct Kit using this configuration. Contact your sales or support representative to obtain a copy of the 3730 DNA Analyzer Human Identification Validation Report. Prepare samples for electrophoresis on the 3730 instrument Prepare the samples for electrophoresis immediately before loading. 1. Calculate the volume of Hi-Di Formamide and size standard needed to prepare the samples: Reagent Volume per reaction Reagent Volume per reaction GeneScan 500 LIZ Size Standard 0.3 µl OR GeneScan 600 LIZ Size Standard v µl Hi-Di Formamide 8.7 µl Hi-Di Formamide 8.5 µl Note: Include additional samples in your calculations to provide excess volume for the loss that occurs during reagent transfers. IMPORTANT! The volume of size standard indicated in the table is a suggested amount. Determine the appropriate amount of size standard based on your experiments and results. AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide 33

34 3 Chapter Instrument Prepare samples for electrophoresis on the 3730 instrument 2. Pipet the required volumes of components into an appropriately sized polypropylene tube. 3. Vortex the tube, then centrifuge briefly. 4. Into each well of a MicroAmp Optical 96-Well Reaction Plate, add: 9 µl of the formamide:size standard mixture 1 µl of PCR product or Allelic Ladder Note: For blank wells, add 10 µl of Hi-Di Formamide. 5. Seal the reaction plate with appropriate septa, then briefly vortex and centrifuge the plate to ensure that the contents of each well are mixed and collected at the bottom. 6. Heat the plate in a thermal cycler for 3 minutes at 95 C. 7. Immediately place the plate on ice for 3 minutes. 8. Place the sample tray on the autosampler. 9. Start the electrophoresis run. 34 AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide

35 4 Analyze Data Section 4.1 GeneMapper ID Software Overview of GeneMapper ID Software Set up GeneMapper ID Software for data analysis Analyze and edit sample files with GeneMapper ID Software Examine and edit a project For more information Section 4.2 GeneMapper ID-X Software Overview of GeneMapper ID-X Software Set up GeneMapper ID-X Software for data analysis Analyze and edit sample files with GeneMapper ID-X Software Examine and edit a project For more information Section 4.1 GeneMapper ID Software Overview of GeneMapper ID Software GeneMapper ID Software is an automated genotyping software for forensic casework, databasing, and paternity data analysis. After electrophoresis, the Data Collection Software stores information for each sample in an.fsa file. Using GeneMapper ID Software v3.2.1 software, you can then analyze and interpret the data from the.fsa files. Instruments Before you start Refer to Instrument and software overview on page 13 for a list of compatible instruments. When using GeneMapper ID Software v3.2.1 to perform human identification (HID) analysis with AmpFlSTR kits, be aware that: HID analysis requires at least one allelic ladder sample per run folder. Perform the appropriate internal validation studies if you want to use multiple ladder samples in an analysis. For multiple ladder samples, the GeneMapper ID Software calculates allelic bin offsets by using an average of all ladders that use the same panel within a run folder. Allelic ladder samples in an individual run folder are considered to be from a single run. AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide 35

36 4 Chapter 4 Analyze Data Set up GeneMapper ID Software for data analysis When the software imports multiple run folders into a project, only the ladder(s) within their respective run folders are used for calculating allelic bin offsets and subsequent genotyping. Allelic ladder samples must be labeled as Allelic Ladder in the Sample Type column in a project. Failure to apply this setting for ladder samples results in failed analysis. Injections containing the allelic ladder must be analyzed with the same analysis method and parameter values that are used for samples, to ensure proper allele calling. Alleles that are not in the AmpFlSTR Allelic Ladders do exist. Off-ladder (OL) alleles may contain full and/or partial repeat units. An off-ladder allele is an allele that occurs outside the ±0.5-nt bin window of any known allelic ladder allele or virtual bin. Note: If a sample allele peak is called as an off-ladder allele, verify the sample result according to your laboratory s protocol. Set up GeneMapper ID Software for data analysis File names Before using the software for the first time Import panels and bins The file names shown in this section may differ from the file names you see when you download or import files. If you need help determining the correct files to use, contact your local Life Technologies Human Identification representative, or go to To analyze sample files (.fsa) using GeneMapper ID Software v3.2.1 for the first time: Import panels and bins into the Panel Manager, as explained in Import panels and bins on page 36. Create an analysis method, as explained in Create an analysis method on page 40. Create a size standard, as explained in Create a size standard on page 45. Define custom views of analysis tables. Refer to the GeneMapper ID Software Versions 3.1 and 3.2 Human Identification Analysis Tutorial (Part no ) for more information. Define custom views of plots. Refer to the GeneMapper ID Software Versions 3.1 and 3.2 Human Identification Analysis Tutorial (Part no ) for more information. To import the Identifiler Direct Kit panel and bin set from the Life Technologies web site into the GeneMapper ID Software v3.2.1 database: 1. Download and open the file containing panels and bins: a. From the Support menu of select Support Software Downloads, Patches & Updates GeneMapper ID Software v 3.2 Updates & Patches, and download the file Identifiler Direct Analysis Files GMID. b. Unzip the file. 36 AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide

37 Section 4.1 GeneMapper ID Software Set up GeneMapper ID Software for data analysis 4 2. Start the GeneMapper ID Software, then log in with the appropriate user name and password. IMPORTANT! For logon instructions, refer to the GeneMapper ID Software Version 3.1 Human Identification Analysis User Guide (Part no ). 3. Select Tools Panel Manager. 4. Find, then open the folder containing the panels and bins: a. Select Panel Manager in the navigation pane. b. Select File Import Panels to open the Import Panels dialog box. c. Navigate to, then open the Identifiler Direct Analysis Files GMID folder that you unzipped in step 1 on page 36. GeneMapper ID Software 5. Select IdentifilerDirect_GS500_Panels_v1.txt, then click Import. Note: Importing this file creates a new folder in the navigation pane of the Panel Manager, IdentifilerDirect_GS500_v1. This folder contains the panel and associated markers. 6. Import IdentifilerDirect_GS500_Bins_v1.txt: a. Select the IdentifilerDirect_GS500_v1 folder in the navigation pane. b. Select File Import Bin Set to open the Import Bin Set dialog box. c. Navigate to, then open the Identifiler Direct Analysis Files GMID folder. AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide 37

38 4 Chapter 4 Analyze Data Set up GeneMapper ID Software for data analysis d. Select IdentifilerDirect_GS500_Bins_v1.txt, then click Import. Note: Importing this file associates the bin set with the panels in the IdentifilerDirect_GS500_Panels_v1 folder. 7. View the imported panels in the navigation pane: a. Double-click the IdentifilerDirect_GS500_v1 folder to view the IdentifilerDirect_GS500_Panels_v1 folder. b. Double-click the IdentifilerDirect_GS500_Panels_v1 folder to display the panel information in the right pane. 38 AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide

39 Section 4.1 GeneMapper ID Software Set up GeneMapper ID Software for data analysis 4 8. Select D8S1179 to display the Bin view for the marker in the right pane. GeneMapper ID Software 9. Click Apply, then OK to add the Identifiler Direct Kit panel and bin set to the GeneMapper ID Software database. IMPORTANT! If you close the Panel Manager without clicking OK, the panels and bins are not imported into the GeneMapper ID Software database. AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide 39

40 4 Chapter 4 Analyze Data Set up GeneMapper ID Software for data analysis Create an analysis method To create an HID analysis method for the Identifiler Direct Kit. 1. Select Tools GeneMapper Manager to open the GeneMapper Manager. 2. Select the Analysis Methods tab, then click New to open the New Analysis Method dialog box. 3. Select HID and click OK to open the Analysis Method Editor with the General tab selected. 4. Enter the settings shown in the figures on the following pages. Note: The Analysis Method Editor closes when you save your settings. To complete this step quickly, do not save the analysis method until you finish entering settings in all of the tabs. 5. After you enter settings in all tabs, click Save. General tab settings 40 AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide

41 Section 4.1 GeneMapper ID Software Set up GeneMapper ID Software for data analysis 4 In the Name field, either type the name as shown for consistency with files supplied with other AmpFlSTR kits, or enter a name of your choosing. The Description and Instrument fields are optional. Allele tab settings GeneMapper ID Software In the Bin Set field, select the IdentifilerDirect_GS500_Bins_v1 bin set imported previously and configure the stutter distance parameters as shown. GeneMapper ID Software v3.2.1 allows you to specify four types of marker repeat motifs: tri, tetra, penta, and hexa. You can enter parameter values for each type of repeat in the appropriate column. Specify the stutter ratio: To apply the stutter ratios listed in the Allele tab for single-source data, deselect the Use marker-specific stutter ratio if available check box (selected by default). Perform appropriate internal validation studies to determine the appropriate filter setting to use. Note: Applying global stutter ratios may reduce the editing required for single-source sample data. To apply the stutter ratios contained in the IdentifilerDirect_GS500_Panels_v1 file, select the Use marker-specific stutter ratio if available check box (selected by default). Perform appropriate internal validation studies to determine the appropriate filter setting to use. AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide 41

42 4 Chapter 4 Analyze Data Set up GeneMapper ID Software for data analysis Peak Detector tab settings Perform internal validation studies to determine settings IMPORTANT! Perform the appropriate internal validation studies to determine the peak amplitude thresholds for interpretation of Identifiler Direct Kit data. Fields include: Peak amplitude thresholds The software uses these parameters to specify the minimum peak height, in order to limit the number of detected peaks. Although GeneMapper ID Software displays peaks that fall below the specified amplitude in electropherograms, the software does not label or determine the genotype of these peaks. Size calling method The Identifiler Direct Kit has been validated using the Local Southern sizing method. Before using alternative sizing methods, perform the appropriate internal validation studies. 42 AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide

43 Section 4.1 GeneMapper ID Software Set up GeneMapper ID Software for data analysis /3500xL and 3730 data 3500/3500xL and 3730 data: Overall peak heights for the data are approximately 3 times higher than peak heights obtained for samples run on the 31xx series instruments. Evaluate validation data carefully to determine the appropriate Peak Amplitude Thresholds for reliable analysis data only: Due to differences in the resolution of peaks using POP-7 polymer versus POP-4 polymer, reduce the Peak Window Size setting in GeneMapper ID Software from 15 pts to 11 pts to obtain accurate genotyping results. For more information: Refer to User Bulletin: Applied Biosystems 3500/3500xL Genetic Analyzer: Protocols for Analysis of AmpFlSTR PCR Amplification Kit PCR Products and Validation Summary (Part no ) Contact your sales or support representative to obtain a copy of the 3730 DNA Analyzer Human Identification Validation Report GeneMapper ID Software Peak Quality tab settings Perform internal validation studies to determine settings IMPORTANT! Perform the appropriate internal validation studies to determine the minimum heterozygous and homozygous minimum peak height thresholds and the minimum peak height ratio threshold that allow for reliable interpretation of Identifiler Direct Kit data. AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide 43

44 4 Chapter 4 Analyze Data Set up GeneMapper ID Software for data analysis Quality Flags tab settings IMPORTANT! The values shown are the software defaults and are the values we used during developmental validation. Perform the appropriate internal validation studies to determine the appropriate values to use in your laboratory. 44 AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide

45 Section 4.1 GeneMapper ID Software Set up GeneMapper ID Software for data analysis 4 Create a size standard The size standards for the Identifiler Direct Kit use the following size standard peaks in their definitions: GeneScan 500 LIZ Size Standard peak sizes GeneScan 600 LIZ Size Standard v2.0 peak sizes 75, 100, 139, 150, 160, 200, 300, 350, 400, and 450 Note: The 250-nt and the 340-nt peak in the GeneScan 500 LIZ Size Standard are not included in the size standard definition. These peaks can be used as an indicator of precision within a run. To create the size standard for the Identifiler Direct Kit: 80, 100, 114, 120, 140, 160, 180, 200, 214, 220, 240, 250, 260, 280, 300, 314, 320, 340, 360, 380, 400, 414, 420, 440 and Select Tools GeneMapper Manager to open the GeneMapper Manager. GeneMapper ID Software 2. Select the Size Standards tab, then click New. AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide 45

46 4 Chapter 4 Analyze Data Analyze and edit sample files with GeneMapper ID Software 3. Enter a name as shown below or enter a name of your choosing. In the Size Standard Dye field, select Orange. In the Size Standard Table, enter the peak sizes specified in on page 45. The example below is for the GeneScan 500 LIZ Size Standard. Analyze and edit sample files with GeneMapper ID Software 1. In the Project window, select File Add Samples to Project, then navigate to the disk or directory containing the sample files. 2. Apply analysis settings to the samples in the project. The names of the settings shown are the names suggested in the sections above. If you named the settings differently, select the names you specified. Parameter Sample Type Analysis Method Panel Size Standard Select the sample type. Settings IdentifilerDirect_AnalysisMethod_v1 (or the name of the analysis method you created) IdentifilerDirect_GS500_Panels_v1 CE_G5_IdentifilerDirect_GS500 (or the name of the size standard you created) For more information about how the Size Caller works, refer to the ABI PRISM GeneScan Analysis Software for the Windows NT Operating System Overview of the Analysis Parameters and Size Caller User Bulletin (Part no ). For additional information about size standards, refer to the GeneMapper ID Software Version 3.1 Human Identification Analysis User Guide (Part no ). 46 AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide

47 Section 4.1 GeneMapper ID Software Examine and edit a project 4 3. Click (Analyze), enter a name for the project (in the Save Project dialog box), then click OK to start analysis. During a run: The status bar displays the progress of analysis as both: A completion bar extending to the right with the percentage completed indicated With text messages on the left The table displays the row of the sample currently being analyzed in green (or red if analysis failed for the sample). The Genotypes tab becomes available after analysis. GeneMapper ID Software Examine and edit a project You can display electropherogram plots from the Samples and Genotypes tabs of the Project window to examine the data. These procedures start with the Samples tab of the Project window (assuming the analysis is complete). For more information For details about GeneMapper ID Software features, allele filters, peak detection algorithms, and project editing, refer to: GeneMapper ID Software Versions 3.1 and 3.2 Human Identification Analysis Tutorial (Part no ) GeneMapper ID Software Version 3.1 Human Identification Analysis User Guide (Part no ) Installation Procedures and New Features for GeneMapper ID Software Software Version v3.2 User Bulletin (Part no ) AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide 47

48 4 Chapter 4 GeneMapper ID-X Software Overview of GeneMapper ID-X Software Section 4.2 GeneMapper ID-X Software Overview of GeneMapper ID-X Software GeneMapper ID-X Software is an automated genotyping software for forensic casework, databasing, and paternity data analysis. After electrophoresis, the data collection software stores information for each sample in a.fsa or.hid file. Using GeneMapper ID-X Software, you can then analyze and interpret the data from the.fsa files (GeneMapper ID-X Software v1.0.1 or higher) or.hid files (GeneMapper ID-X Software v1.2 or higher). Instruments Before you start Refer to Instrument and software overview on page 13 for a list of compatible instruments. When using GeneMapper ID-X Software v1.0.1 or higher to perform human identification (HID) analysis with AmpFlSTR kits, be aware that: HID analysis requires at least one allelic ladder sample per run folder. Perform the appropriate internal validation studies if you want to use multiple ladder samples in an analysis. For multiple ladder samples, the GeneMapper ID-X Software calculates allelic bin offsets by using an average of all ladders that use the same panel within a run folder. Allelic ladder samples in an individual run folder are considered to be from a single run. When the software imports multiple run folders into a project, only the ladder(s) within their respective run folders are used for calculating allelic bin offsets and subsequent genotyping. Allelic ladder samples must be labeled as Allelic Ladder in the Sample Type column in a project. Failure to apply this setting for ladder samples results in failed analysis. Injections containing the allelic ladder must be analyzed with the same analysis method and parameter values that are used for samples to ensure proper allele calling. Alleles that are not in the AmpFlSTR Allelic Ladders do exist. Off-ladder (OL) alleles may contain full and/or partial repeat units. An off-ladder allele is an allele that occurs outside the ±0.5-nt bin window of any known allelic ladder allele or virtual bin. Note: If a sample allele peak is called as an off-ladder allele, verify the sample result according to your laboratory protocol. 48 AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide

49 Section 4.2 GeneMapper ID-X Software Set up GeneMapper ID-X Software for data analysis 4 Set up GeneMapper ID-X Software for data analysis File names The file names shown in this section may differ from the file names you see when you download or import files. If you need help determining the correct files to use, contact your local Life Technologies Human Identification representative, or go to Before using the software for the first time Before you use GeneMapper ID-X Software to analyze data files (GeneMapper ID-X Software v1.0.1 or higher for.fsa files, GeneMapper ID-X Software v1.2 or higher for.hid files): Import panels, bins, and marker stutter into the Panel Manager, as explained in Import panels, bins, and marker stutter on page 49. Create an analysis method, as explained in Create an analysis method on page 53. Create a size standard, as explained in Create a size standard on page 58. Define custom views of analysis tables. Refer to the GeneMapper ID-X Software Version 1.0 Getting Started Guide (Part no ) for more information. Define custom views of plots. Refer to the GeneMapper ID-X Software Version 1.0 Getting Started Guide (Part no ) for more information. GeneMapper ID-X Software Import panels, bins, and marker stutter To import the Identifiler Direct Kit panel, bin set, and marker stutter from the Life Technologies web site into the GeneMapper ID-X Software database: 1. Download and open the file containing panels, bins, and marker stutter: a. From the Support menu of select Support Software Downloads, Patches & Updates GeneMapper ID-X Software Updates & Patches, and download the file Identifiler Direct Analysis Files GMIDX. b. Unzip the file. 2. Start the GeneMapper ID-X Software, then log in with the appropriate user name and password. IMPORTANT! For logon instructions, refer to the GeneMapper ID-X Software Version 1.0 Getting Started Guide (Part no ). 3. Select Tools Panel Manager. 4. Find, then open the folder containing the panels, bins, and marker stutter: a. Select Panel Manager in the navigation pane. b. Select File Import Panels to open the Import Panels dialog box. c. Navigate to, then open the Identifiler Direct Analysis Files GMIDX folder that you unzipped in step 1. AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide 49

50 4 Chapter 4 GeneMapper ID-X Software Set up GeneMapper ID-X Software for data analysis 5. Select IdentifilerDirect_GS500_v1X, then click Import. Note: Importing this file creates a new folder in the navigation pane of the Panel Manager IdentifilerDirect_GS500_Panels_v1X. This folder contains the panel and associated markers. 6. Import IdentifilerDirect_GS500_Bins_v1X.txt: a. Select the IdentifilerDirect_GS500_Panels_v1X folder in the navigation pane. b. Select File Import Bin Set to open the Import Bin Set dialog box. c. Navigate to, then open the Identifiler Direct Analysis Files GMIDX folder. d. Select IdentifilerDirect_GS500_Bins_v1X.txt, then click Import. Note: Importing this file associates the bin set with the panels in the IdentifilerDirect_GS500_v1X folder. 50 AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide

51 Section 4.2 GeneMapper ID-X Software Set up GeneMapper ID-X Software for data analysis 4 7. View the imported panels in the navigation pane: a. Double-click the IdentifilerDirect_GS500_v1X folder. b. Double-click the IdentifilerDirect_GS500_Panels_v1X folder to display the panel information in the right pane and the markers below it. GeneMapper ID-X Software 8. Select D8S1179 to display the Bin view for the marker in the right pane. AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide 51

52 4 Chapter 4 GeneMapper ID-X Software Set up GeneMapper ID-X Software for data analysis 9. Import IdentifilerDirect_GS500_Stutter_v1X.txt: a. Select the IdentifilerDirect_GS500_v1 folder in the navigation panel. b. Select File Import Marker Stutter to open the Import Marker Stutter dialog box. c. Navigate to, then open the Identifiler Direct Analysis Files GMIDX folder. d. Select IdentifilerDirect_GS500_Stutter_v1X.txt, then click Import. Note: Importing this file associates the marker stutter ratio with the bin set in the IdentifilerDirect_GS500_Bins_v1X folder. 10. View the imported marker stutters in the navigation pane: a. Select the IdentifilerDirect_GS500_v1X folder to display its list of markers in the right pane. b. Double-click the IdentifilerDirect_GS500_v1X folder to display its list of markers below it. 52 AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide

53 Section 4.2 GeneMapper ID-X Software Set up GeneMapper ID-X Software for data analysis 4 c. Double-click D16S539 to display the Stutter Ratio & Distance view for the marker in the right pane. GeneMapper ID-X Software 11. Click Apply, then OK to add the Identifiler Direct Kit panel, bin set, and marker stutter to the GeneMapper ID-X Software database. IMPORTANT! If you close the Panel Manager without clicking Apply, the panels, bin sets, and marker stutter will not be imported into the GeneMapper ID-X Software database. Create an analysis method Use the following procedure to create an analysis method for the Identifiler Direct Kit. IMPORTANT! Analysis methods are version-specific, so you must create an analysis method for each version of the software. For example, an analysis method created for GeneMapper ID-X Software version 1.2 is not compatible with earlier versions of GeneMapper ID-X Software, or with GeneMapper ID Software version Select Tools GeneMapper ID-X Manager to open the GeneMapper ID-X Manager. AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide 53

54 4 Chapter 4 GeneMapper ID-X Software Set up GeneMapper ID-X Software for data analysis 2. Select the Analysis Methods tab, then click New to open the Analysis Method Editor with the General tab selected. 3. Enter the settings shown in the figures on the following pages. Note: The Analysis Method Editor closes when you save your settings. To complete this step quickly, do not save the analysis method until you finish entering settings in all of the tabs. 4. After you enter the settings on all tabs, click Save. General tab settings In the Name field, either type the name as shown or enter a name of your choosing. In the Security Group field, select the Security Group appropriate to your software configuration from the drop-down list. The Description and Instrument fields are optional. 54 AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide

55 Section 4.2 GeneMapper ID-X Software Set up GeneMapper ID-X Software for data analysis 4 Allele tab settings GeneMapper ID-X Software In the Bin Set field, select the IdentifilerDirect_GS500_Bins_v1X bin set imported previously and configure the parameters as shown. GeneMapper ID-X Software allows you to specify 4 types of marker repeat motifs: tri, tetra, penta and hexa. You can enter parameter values for each type of repeat in the appropriate column. Specify the stutter ratio: To apply the stutter ratios listed in the Allele tab for single-source data, deselect the Use marker-specific stutter ratio if available check box (selected by default). Perform appropriate internal validation studies to determine the appropriate filter setting to use. Note: Applying global stutter ratios may reduce the editing required for single-source sample data. To apply the stutter ratios contained in the IdentifilerDirect_GS500_Stutter_v1X.txt file, select the Use marker-specific stutter ratio if available check box (selected by default). Perform appropriate internal validation studies to determine the appropriate filter setting to use. AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide 55

56 4 Chapter 4 GeneMapper ID-X Software Set up GeneMapper ID-X Software for data analysis Peak Detector tab settings Perform internal validation studies to determine settings IMPORTANT! Perform the appropriate internal validation studies to determine the appropriate peak amplitude thresholds for interpretation of Identifiler Direct Kit data. Fields include: Peak amplitude thresholds The software uses these parameters to specify the minimum peak height, in order to limit the number of detected peaks. Although GeneMapper ID-X Software displays peaks that fall below the specified amplitude in electropherograms, the software does not label or determine the genotype of these peaks. Size calling method This kit has been validated using the Local Southern sizing method. Before using alternative sizing methods, perform the appropriate internal validation studies. Normalization (v1.2 or higher) For use with 3500 data. Perform internal validation studies to determine whether to use the Normalization feature for analysis of Identifiler Direct Kit data. 56 AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide

57 Section 4.2 GeneMapper ID-X Software Set up GeneMapper ID-X Software for data analysis 4 Peak Quality tab settings Perform internal validation studies to determine settings GeneMapper ID-X Software IMPORTANT! Perform the appropriate internal validation studies to determine the minimum heterozygous and homozygous minimum peak height thresholds, maximum peak height threshold, and the minimum peak height ratio threshold for interpretation of Identifiler Direct Kit data. AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide 57

58 4 Chapter 4 GeneMapper ID-X Software Set up GeneMapper ID-X Software for data analysis SQ & GQ tab settings IMPORTANT! The values shown are the software defaults and are the values we used during developmental validation. Perform appropriate internal validation studies to determine the appropriate values to use. Create a size standard The size standards for the Identifiler Direct Kit uses the following size standard peaks in their definitions: GeneScan 500 LIZ Size Standard peak sizes 75, 100, 139, 150, 160, 200, 300, 350, 400, and 450 GeneScan 600 LIZ Size Standard v2.0 peak sizes 80, 100, 114, 120, 140, 160, 180, 200, 214, 220, 240, 250, 260, 280, 300, 314, 320, 340, 360, 380, 400, 414, 420, 440 and 460 Note: The 250-nt and the 340-nt peaks in the GeneScan 500 LIZ Size Standard are not included in the size standard definition. These peaks can be used as an indicator of precision within a run. Use the following procedure to create the size standard for the Identifiler Direct Kit. 1. Select Tools GeneMapper ID-X Manager to open the GeneMapper ID-X Manager. 58 AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide

59 Section 4.2 GeneMapper ID-X Software Set up GeneMapper ID-X Software for data analysis 4 2. Select the Size Standards tab, then click New. 3. Complete the Name field as shown below or with a name of your choosing. In the Security Group field, select the Security Group appropriate to your software configuration from the drop-down list. In the Size Standard Dye field, select Orange. In the Size Standard Table, enter the peak sizes specified on page 58. The example below is for the GeneScan 500 LIZ Size Standard. GeneMapper ID-X Software AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide 59

60 4 Chapter 4 GeneMapper ID-X Software Analyze and edit sample files with GeneMapper ID-X Software Analyze and edit sample files with GeneMapper ID-X Software 1. In the Project window, select File Add Samples to Project, then navigate to the disk or directory containing the sample files. 2. Apply analysis settings to the samples in the project. The names of the settings shown are the names suggested in the sections above. If you named the settings differently, select the names you specified. Parameter Sample Type Analysis Method Panel Size Standard Settings Select the sample type. IdentifilerDirect_AnalysisMethod_v1X (or the name of the analysis method you created) IdentifilerDirect_GS500_Panels_v1X CE_G5_IdentifilerDirect_GS500 (or the name of the size standard you created) For more information about how the Size Caller works, or about size standards, refer to the GeneMapper ID-X Software v1.2 Reference Guide (Part no ). 3. Click (Analyze), enter a name for the project (in the Save Project dialog box), then click OK to start analysis. During a run: The status bar displays the progress of analysis as a completion bar extending to the right with the percentage completed indicated. The table displays the row of the sample currently being analyzed in green (or red if analysis failed for the sample). The Analysis Summary tab is displayed upon completion of the analysis. The figure below shows the analysis summary window after analysis. 60 AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide

61 Section 4.2 GeneMapper ID-X Software Examine and edit a project 4 Examine and edit a project You can display electropherogram plots from the Samples and Genotypes tabs of the Project window to examine the data. These procedures start with the Analysis Summary tab of the Project window (assuming the analysis is complete). For more information For more information about any of these tasks, refer to: GeneMapper ID-X Software Version 1.0 Getting Started Guide (Part no ) GeneMapper ID-X Software Version 1.0 Quick Reference Guide (Part no ) GeneMapper ID-X Software Version 1.0 Reference Guide (Part no ) GeneMapper ID-X Software Version 1.1(Mixture Analysis) Getting Started Guide (Part no ) GeneMapper ID-X Software Version 1.2 Reference Guide (Part no ) GeneMapper ID-X Software Version 1.2 Quick Reference Guide (Part no ) GeneMapper ID-X Software AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide 61

62 4 Chapter 4 GeneMapper ID-X Software For more information 62 AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide

63 5 Experiments and Results Overview Accuracy, precision, and reproducibility Extra peaks in the electropherogram Characterization of loci Species specificity Sensitivity Stability Population data Mutation rate Probability of identity Probability of paternity exclusion Overview This chapter provides results of the developmental validation experiments we performed using the Identifiler Direct Kit for samples punched from FTA cards. Importance of validation Experiment conditions Validation of a DNA typing procedure for human identification applications is an evaluation of the procedure s efficiency, reliability, and performance characteristics. By challenging the procedure with samples commonly encountered in forensic and parentage laboratories, the validation process uncovers attributes and limitations which are critical for sound data interpretation in casework (Sparkes, Kimpton, Watson et al., 1996; Sparkes, Kimpton, Gilbard et al., 1996; Wallin et al., 1998). We performed experiments to evaluate the performance of the Identifiler Direct Kit. according to the DNA Advisory Board (DAB) Quality Assurance Standards, effective October 1, 1998 (DNA Advisory Board, 1998). The DAB standards describe the quality assurance requirements that a laboratory should follow to ensure the quality and integrity of the data and competency of the laboratory. Additional validation was performed according to the revised guidelines from the Scientific Working Group on DNA Analysis Methods (SWGDAM, July 10, 2003). Based on these guidelines, we conducted experiments that comply with guidelines 1.0 and 2.0 and its associated subsections. This DNA methodology is not novel. (Moretti et al., 2001; Frank et al., 2001; Wallin et al., 2002; and Holt et al., 2000). AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide 63

64 5 Chapter 5 Experiments and Results Accuracy, precision, and reproducibility This chapter discusses many of the experiments we performed and provides examples of results obtained. We chose conditions that produced optimum PCR product yield and that met reproducible performance standards. It is our opinion that while these experiments are not exhaustive, they are appropriate for a manufacturer of STR kits intended for forensic and/or parentage testing use. Each laboratory using the Identifiler Direct Kit should perform their own internal validation studies. Validation studies included testing on the following sample+substrate combinations: Unpurified, single-source blood or buccal samples on FTA paper (treated paper substrate) Buccal samples on a Bode Buccal DNA Collector (untreated paper substrate) Additional performance verification studies included testing on Copan 4N6FLOQSwabs (swab substrate). We did not perform mixture or inhibition studies during the developmental validation of the Identifiler Direct Kit because these tests are not relevant for the intended use of this chemistry. Accuracy, precision, and reproducibility SWGDAM guideline SWGDAM guideline 2.9 Accuracy Developmental validation is the demonstration of the accuracy, precision, and reproducibility of a procedure by the manufacturer, technical organization, academic institution, government laboratory, or other party. (SWGDAM, July 2003) The extent to which a given set of measurements of the same sample agree with their mean and the extent to which these measurements match the actual values being measured should be determined. (SWGDAM, July 2003) Laser-induced fluorescence detection of length polymorphism at short tandem repeat loci is not a novel methodology (Holt et al., 2000; and Wallin et al., 2002). However, accuracy and reproducibility of Identifiler Direct Kit profiles have been determined from various sample types. Figure 3 illustrates the size differences that are typically observed between sample alleles and allelic ladder alleles on the Applied Biosystems 3130xl Genetic Analyzer with POP-4 polymer. The x-axis in Figure 3 represents the nominal nucleotide sizes for the AmpFlSTR Identifiler Direct Allelic Ladder. The dashed lines parallel to the x-axis represent ±0.25-nt windows. The y-axis represents the deviation of each sample allele size from the corresponding allelic ladder allele size. All sample alleles are within ±0.5 nt from a corresponding allele in the allelic ladder. 64 AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide

65 Chapter 5 Experiments and Results Accuracy, precision, and reproducibility 5 Figure 3 Size deviation of 200 blood samples on FTA card analyzed on the Applied Biosystems 3130xl Genetic Analyzer Precision and size windows Sizing precision allows for determining accurate and reliable genotypes. Sizing precision was measured on the Applied Biosystems 3130xl Genetic Analyzer. The recommended method for genotyping is to employ a ±0.5-nt window around the size obtained for each allele in the AmpFlSTR Identifiler Direct Allelic Ladder. A ±0.5-nt window allows for the detection and correct assignment of alleles. Any sample allele that sizes outside the specified window could be: An off-ladder allele, that is, an allele of a size that is not represented in the AmpFlSTR Identifiler Direct Allelic Ladder or An allele that corresponds to an allelic ladder allele, but whose size falls just outside a window because of measurement error The measurement error inherent in any sizing method can be defined by the degree of precision in sizing an allele multiple times. Precision is measured by calculating the standard deviation in the size values obtained for an allele that is run in several injections on a capillary instrument. Table 3 on page 66 shows typical precision results obtained from five runs (16 capillaries/run) of the AmpFlSTR Identifiler Direct Allelic Ladder on the Applied Biosystems 3130xl Genetic Analyzer (36-cm capillary and POP-4 polymer) sized using the GeneScan 500 LIZ Size Standard. The results were obtained within a consecutive set of injections on a single capillary array. Sample alleles may occasionally size outside of the ±0.5-nt window for a respective allelic ladder allele because of measurement error. The frequency of such an occurrence is lowest in detection systems having the smallest standard deviations in sizing. Figure 3 illustrates the tight clustering of allele sizes obtained on the Applied AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide 65

66 5 Chapter 5 Experiments and Results Accuracy, precision, and reproducibility Biosystems 3130xl Genetic Analyzer, where the standard deviation in sizing is typically less than 0.15 nt. The instance of a sample allele sizing outside the ±0.5-nt window because of measurement error is relatively rare when the standard deviation in sizing is approximately 0.15 nt or less (Smith, 1995). For sample alleles that do not size within a ±0.5-nt window, the PCR product must be rerun to distinguish between a true off-ladder allele versus measurement error of a sample allele that corresponds with an allele in the allelic ladder. Repeat analysis, when necessary, provides an added level of confidence to the final allele assignment. GeneMapper ID Software and GeneMapper ID-X Software automatically flag sample alleles that do not size within the prescribed window around an allelic ladder allele by labelling the allele as OL (Off-ladder). Maximum precision is obtained with a set of capillary injections on each of the supported platforms however the determined allele sizes will vary between the different platforms. Cross-platform sizing differences occur from a number of factors including type and concentration of polymer, run temperature, and electrophoresis conditions. Variations in sizing can also occur between runs on the same instrument and between runs on different instruments of the same platform type because of these factors. We recommend strongly that the allele sizes obtained should be compared to the sizes obtained for known alleles in the AmpFlSTR Identifiler Direct Allelic Ladder from the same run and then converted to genotypes (as described in Before you start on page 35 and 48). Refer to Table 3 for the results of five runs of the AmpFlSTR Identifiler Direct Allelic Ladder. For more information on precision and genotyping, see Lazaruk et al., 1998 and Mansfield et al., In Table 3, the mean sizes for all the alleles in each run (16 capillaries) were calculated. The mean range shown in the table represents the lowest- and highest-mean size values obtained across all five runs. Similarly, the standard deviation for the allele sizing was calculated for all the alleles in each run. The standard deviation range shown in Table 3 represents the lowest and highest standard deviation values obtained across all five runs. Table 3 Precision results of five runs (16 capillaries/run) of the AmpFlSTR Identifiler Direct Allelic Ladder Applied Biosystems 3130xl Genetic Analyzer Allele Mean Standard deviation Amelogenin X Y CSF1PO AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide

67 Chapter 5 Experiments and Results Accuracy, precision, and reproducibility 5 Applied Biosystems 3130xl Genetic Analyzer Allele Mean Standard deviation D13S D16S D18S AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide 67

68 5 Chapter 5 Experiments and Results Accuracy, precision, and reproducibility Applied Biosystems 3130xl Genetic Analyzer Allele Mean Standard deviation D19S D21S AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide

69 Chapter 5 Experiments and Results Accuracy, precision, and reproducibility 5 Applied Biosystems 3130xl Genetic Analyzer Allele Mean Standard deviation D2S AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide 69

70 5 Chapter 5 Experiments and Results Accuracy, precision, and reproducibility Applied Biosystems 3130xl Genetic Analyzer Allele Mean Standard deviation D3S1358 D5S818 D7S820 D8S AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide

71 Chapter 5 Experiments and Results Accuracy, precision, and reproducibility 5 Applied Biosystems 3130xl Genetic Analyzer Allele Mean Standard deviation FGA AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide 71

72 5 Chapter 5 Experiments and Results Accuracy, precision, and reproducibility Applied Biosystems 3130xl Genetic Analyzer Allele Mean Standard deviation TH01 TPOX vwa AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide

73 Chapter 5 Experiments and Results Extra peaks in the electropherogram 5 Extra peaks in the electropherogram Causes of extra peaks Stutter products Peaks other than the target alleles may be detected on the electropherogram. Causes for the appearance of extra peaks include stutter products, incomplete 3 A nucleotide addition (at the n-1 position), dye artifacts, and mixed DNA samples (see DAB Standard ). A stutter is a well-characterized PCR artifact that refers to the appearance of a minor peak one repeat unit smaller (or less frequently, one repeat larger) than the major STR product (Butler, 2005; Mulero et al., 2006). Sequence analysis of stutter products at tetranucleotide STR loci has revealed that the stutter product is missing a single tetranucleotide core repeat unit relative to the main allele (Walsh et al., 1996). The proportion of the stutter product relative to the main allele (percent stutter) is measured by dividing the height of the stutter peak by the height of the main allele peak. Peak heights were measured for amplified samples at the loci used in the Identifiler Direct Kit: Treated paper workflow: 370 blood samples on FTA card and 299 buccal samples on Indicating FTA cards Untreated paper workflow: 370 buccal samples on Bode Buccal DNA Collectors All data were generated on the Applied Biosystems 3130xl Genetic Analyzer. Some conclusions from these measurements and observations are: For each Identifiler Direct Kit locus, the percent stutter generally increases with allele length, as shown in Treated paper workflow: Figure 4 through Figure 7 on page 74 through page 75 Untreated paper workflow: Figure 8 through Figure 11 on page 76 through page 77 Smaller alleles display a lower level of stutter relative to the longer alleles within each locus. Each allele within a locus displays a percent stutter that is consistent. The stutter value for each locus shown for the treated paper workflow in Table 4 on page 78 was determined by taking the mean plus three times the standard deviation. These values are the stutter filter percentages in the Identifiler Direct stutter file and will be used during the filtering step in GeneMapper ID Software or GeneMapper ID-X Software. Peaks in the stutter position that are above the stutter filter percentage will not be filtered. Peaks in the stutter position that have not been filtered and remain labeled can be further evaluated. Stutter percentages generated using the untreated paper workflow were calculated on a different, smaller data set than was used for the original stutter calculations. We used the stutter values of the most common alleles at each locus to compare the data sets. There was no significant difference in the stutter values (mean plus three times the standard deviation) for the individual loci with the exception of D3S1358 (0.7%), D7S820 (0.4%), D16S539 (0.2%) and FGA (1.2%). For D3S1358, D7S820, and FGA, the stutter values were slightly lower than the original stutter values calculated for punches from FTA cards processed with the AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide 73

74 5 Chapter 5 Experiments and Results Extra peaks in the electropherogram Identifiler Direct Kit. The D16S539 stutter percentage (mean plus three times the standard deviation) was slightly higher than the original stutter value. You should evaluate the impact of sample type on stutter percentages when implementing a direct amplification system. The measurement of percent stutter for allele peaks that are off-scale may be unusually high. Off-scale peaks were not included in the evaluation of stutter characterized here. Figure 4 Treated paper workflow: FTA card sample stutter percentages for D8S1179, D21S11, D7S820, and CSF1PO loci (red = blood samples; blue = buccal samples) Figure 5 Treated paper workflow: FTA card sample stutter percentages for D3S1358, TH01, D13S317, D16S539, and D2S1338 loci (red = blood samples; blue = buccal samples) 74 AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide

75 Chapter 5 Experiments and Results Extra peaks in the electropherogram 5 Figure 6 Treated paper workflow: FTA card sample stutter percentages for D19S433, vwa, TPOX, and D18S51 loci (red = blood samples; blue = buccal samples) Figure 7 Treated paper workflow: FTA card sample stutter percentages for D5S818 and FGA loci (red = blood samples; blue = buccal samples) AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide 75

76 5 Chapter 5 Experiments and Results Extra peaks in the electropherogram Figure 8 Untreated paper workflow: Bode Buccal DNA Collector sample stutter percentages for D8S1179, D21S11, D7S820, and CSF1PO loci Figure 9 Untreated paper workflow: Bode Buccal DNA Collector sample stutter percentages for D3S1358, TH01, D13S317, D16S539, and D2S1338 loci 76 AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide

77 Chapter 5 Experiments and Results Extra peaks in the electropherogram 5 Figure 10 Untreated paper workflow: Bode Buccal DNA Collector sample stutter percentages for D19S433, vwa, TPOX, and D18S51 loci Figure 11 Untreated paper workflow: Bode Buccal DNA Collector sample stutter percentages for D5S818 and FGA loci (red = blood samples; blue = buccal samples) AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide 77

78 5 Chapter 5 Experiments and Results Extra peaks in the electropherogram Table 4 Treated paper workflow: FTA card sample marker-specific stutter filter percentages for Identifiler Direct Kit loci Locus % Stutter CSF1PO 8.48 D13S D16S D18S D19S D21S D2S D3S D5S D7S D8S FGA TH TPOX 5.27 vwa These percentages are used as stutter filters in GeneMapper ID Software IdentifilerDirect_GS500_Panels_v1 and GeneMapper ID-X Software IdentifilerDirect_GS500_Stutter_v1X.txt. Addition of 3 A nucleotide AmpliTaq Gold enzyme, like many other DNA polymerases, can catalyze the addition of a single nucleotide (predominately adenosine) to the 3 ends of doublestranded PCR products (Clark, 1988; Magnuson et al., 1996). This nontemplate addition results in a PCR product that is one nucleotide longer than the actual target sequence. The PCR product with the extra nucleotide is referred to as the +A form. The efficiency of +A addition is related to the particular sequence of the DNA at the 3 end of the PCR product. The AmpFlSTR Identifiler Direct PCR Amplification Kit includes two main design features that promote maximum +A addition: The primer sequences have been optimized to encourage +A addition. The final extension step is 60 C for 25 min. The final extension step gives the AmpliTaq Gold DNA polymerase additional time to complete +A addition to all double-stranded PCR products. STR systems (where each allele is represented by two peaks that are one nucleotide apart) that have not been optimized for +A addition may have split peaks. 78 AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide

79 Chapter 5 Experiments and Results Characterization of loci 5 Figure 12 Omitting the final extension step results in split peaks due to incomplete A nucleotide addition. Data are from an Applied Biosystems 3130xl Genetic Analyzer using the AmpFlSTR Identifiler Direct Kit. Artifacts Artifacts and anomalies are seen in all molecular biological systems. Artifacts are typically reproducible while anomalies are non-reproducible, intermittent occurrences that are not observed consistently in a system (for example, spikes and baseline noise). Reproducible artifacts have not been seen in data produced on the genetic analyzers used during developmental validation of the Identifiler Direct Kit. Characterization of loci SWGDAM guideline 2.1 Nature of the polymorphisms The basic characteristics of a genetic marker must be determined and documented. (SWGDAM, July 2003) This section describes basic characteristics of the 15 loci and the sex-determining marker, Amelogenin that are amplified with the Identifiler Direct Kit. These loci have been extensively characterized by other laboratories. The primers for the Amelogenin locus flank a 6-nucleotide deletion within intron 1 of the X homologue. Amplification results in 107-nt and 113-nt products from the X and Y chromosomes, respectively. (Sizes are the actual nucleotide size according to sequencing results, including 3 A nucleotide addition.) The remaining Identifiler Direct Kit loci are all tetranucleotide short tandem repeat (STR) loci. The length differences among alleles of a particular locus result from differences in the number of 4-nt repeat units. AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide 79

80 5 Chapter 5 Experiments and Results Species specificity We have subjected to sequencing all the alleles in the AmpFlSTR Identifiler Direct Allelic Ladder. In addition, other groups in the scientific community have sequenced alleles at some of these loci. Among the various sources of sequence data on the Identifiler Direct Kit loci, there is consensus on the repeat patterns and structure of the STRs. Mapping The Identifiler Direct Kit loci have been mapped, and the chromosomal locations have been published (Nakahori et al., 1991; Edwards et al., 1992; Kimpton et al., 1992; Mills et al., 1992; Sharma and Litt, 1992; Li et al., 1993; Straub et al., 1993; Barber and Parkin, 1996). Species specificity SWGDAM Guideline 2.2 For techniques designed to type human DNA, the potential to detect DNA from forensically relevant nonhuman species should be evaluated. (SWGDAM, July 2003) The Identifiler Direct Kit provides the required specificity for detecting primate alleles. Other species do not amplify for the loci tested. Nonhuman studies Nonhuman DNA may be present in forensic casework samples. The data from Identifiler Direct Kit experiments on nonhuman DNA sources are shown in Figure 13. Figure 13 Representative electropherograms from a species-specificity study including positive and non-template controls (NTC) Control DNA 9947A Chimpanzee Pig Cat Microbial pool NTC 80 AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide

81 Chapter 5 Experiments and Results Sensitivity 5 Figure 13 shows amplification for: Control DNA 9947A (1 ng, panel 1), chimpanzee (1 ng, panel 2), pig (10 ng, panel 3), cat (10 ng, panel 4), microbial DNA pool (equivalent to 105 copies of Candida albicans, Enterococcus faecalis, Escherichia coli, Fusobacterium nucleatum, Lactobacillus casei, Staphylococcus aureus, Streptococcus mitis, Streptococcus mutans, Streptococcus salivarius, and Streptococcus viridans, panel 5), and the non-template control (panel 6). The extracted DNA samples were amplified with the Identifiler Direct Kit and analyzed using the Applied Biosystems 3130xl Genetic Analyzer. Primates: gorilla, chimpanzee, orangutan, and macaque (1 ng each) Non-primates: mouse, dog, pig, cat, horse, hamster, rat, chicken, and cow (10 ng each) Microorganisms: Candida albicans, Enterococcus faecalis, Escherichia coli, Fusobacterium nucleatum, Lactobacillus casei, Staphylococcus aureus, Streptococcus mitis, Streptococcus mutans, Streptococcus salivarius, and Streptococcus viridans (equivalent to 105 copies). These microorganisms are commonly found in the oral cavity (Suido et al., 1986; Guthmiller et al., 2001). All the primate DNA samples amplified, producing fragments within the 100 to 350 base pair region (Lazaruk, et al., 2001; Wallin, et al., 1998). The microorganisms, chicken, cat, hamster, rat, rabbit, and mouse samples did not yield detectable product. Horse, cow, dog, and pig samples produced a 104-bp fragment near the Amelogenin locus in PET dye. Sensitivity SWGDAM guideline 2.3 Blood on FTA cards Buccal cells on FTA or Indicating FTA cards and buccal cells on Bode DNA Collectors When appropriate, the range of DNA quantities able to produce reliable typing results should be determined. (SWGDAM, July 2003) The Identifiler Direct Kit has been optimized at 25 µl PCR reaction volume to overcome the PCR inhibition expected when amplifying blood samples directly from unpurified 1.2 mm FTA discs. Depending on the volume of blood spotted onto the FTA card, DNA quantities present on the 1.2 mm disc may vary from laboratory to laboratory. It is essential for your laboratory to optimize the PCR conditions based on the types of blood samples received or based on your standard operating protocol used in the spotting of blood onto FTA cards. Refer to page 17 for instructions on PCR optimization. The Identifiler Direct Kit has been optimized at 25 µl PCR reaction volume to overcome the PCR inhibition expected when amplifying buccal cells directly from unpurified 1.2 mm FTA discs or Indicating FTA discs. Depending on the collecting devices used, the collection methods applied, and the swab-to-fta transfer protocol employed, DNA quantities present on the 1.2 mm disc may vary from sample to sample and from laboratory to laboratory. It is essential for your laboratory to optimize the PCR conditions based on the types of buccal samples received or based on your standard operating protocol used in transferring saliva from a buccal swab onto an FTA card or Indicating FTA cards. Refer to page 17 for instructions on PCR optimization. AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide 81

82 5 Chapter 5 Experiments and Results Sensitivity Effect of DNA quantity on results If too much DNA is added to the PCR reaction, the increased amount of PCR product that is generated can result in: Fluorescence intensity that exceeds the linear dynamic range for detection by the instrument ( off-scale data). Off-scale data is a problem because: Quantitation (peak height and area) for off-scale peaks is not accurate. For example, an allele peak that is off-scale can cause the corresponding stutter peak to appear higher in relative intensity, thus increasing the calculated percent stutter. Multicomponent analysis of off-scale data is not accurate. This inaccuracy results in poor spectral separation ( pull-up ). Incomplete +A nucleotide addition. To ensure minimal occurrence of offscale data when using the Identifiler Direct Kit, optimize PCR cycle number according to instructions on page 17. When the total number of allele copies added to the PCR is extremely low, unbalanced amplification of the alleles may occur because of stochastic fluctuation. Individual laboratories may find it useful to determine an appropriate minimum peak height threshold based on their own results and instruments using low amounts of input DNA. 82 AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide

83 Chapter 5 Experiments and Results Stability 5 Figure 14 Effect of amplifying varying amounts of white blood cells (WBCs) spotted onto Indicating FTA discs Note that the y-axis scale is magnified for the lower amounts of DNA, analyzed using the Applied Biosystems 3130xl Genetic Analyzer. The amount of DNA on the Indicating FTA cards were calculated based on the assumptions of 100% cell lysis efficiency and that each cell contain 6 pg of DNA. The results from white blood cells spotted onto Bode DNA Collectors were comparable to the results shown here obtained using the Identifiler Direct Kit with white blood cells spotted onto FTA Indicating Cards (data not shown). Stability SWGDAM guideline 2.4 The ability to obtain results from DNA recovered from biological samples deposited on various substrates and subjected to various environmental and chemical insults has been extensively documented. In most instances, assessment of the effects of these factors on new forensic DNA procedures is not required. However, if substrates and/or environmental and/or chemical insults could potentially affect the analytical process, then the process should be evaluated using known samples to determine the effects of such factors. (SWGDAM, July 2003) AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide 83

84 5 Chapter 5 Experiments and Results Stability DNA on FTA cards Aged blood on FTA cards and aged buccal cells on Indicating FTA cards were prepared to examine the sample-on-substrate stability. Finger-prick blood spotted onto FTA card and buccal samples swabbed and transferred using the EasiCollect devices were collected on three individuals over the course of 30 weeks. The Identifiler Direct Kit was used to amplify the aged FTA samples in a GeneAmp PCR System 9700 with the gold-plated silver 96-well block and were electrophoresed and detected using an Applied Biosystems 3130xl Genetic Analyzer. The results of the aged blood on FTA card are shown in Figure 15 and the results of the aged buccal cells on Indicating FTA card are shown in Figure 16. The analysis revealed that the age of the FTA samples did not impact the performance of the AmpFlSTR Identifiler Direct Kit. Figure 15 Amplification of blood on FTA card stored for various amounts of time at room temperature Figure 16 Amplification of buccal cells on Indicating FTA card stored for various amounts of time at room temperature 84 AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide

85 Chapter 5 Experiments and Results Stability 5 DNA on buccal swabs Aged buccal cell samples on Copan 4N6FLOQSwabs, Whatman OmniSwabs, and Puritan swabs were also prepared to verify their respective sample-on-substrate stability. Buccal swabs were collected from 40 individuals on each swab type over the course of three months. The aged swab samples were processed with Prep-n-Go Buffer, amplified using the Identifiler Direct Kit in a GeneAmp PCR System 9700 with the gold-plated silver 96-well block, and were electrophoresed and detected using an Applied Biosystems 3130xl Genetic Analyzer. Figure 17 shows the results of the aged buccal samples collected on each swab type and lysed at 90 C for 20 minutes. For comparison, Figure 18 shows the results of fresh buccal samples collected on Copan 4N6FLOQSwabs and lysed at room temperature. The analysis revealed that buccal samples on the swab types tested, air-dried immediately after collection, and aged up to three months at room temperature produce acceptable profiles when amplified with the Identifiler Direct Kit. Figure 17 Amplification of buccal cells on aged Copan 4N6FLOQSwabs, OmniSwabs, and Puritan swabs stored for 3 months at room temperature and lysed with Prep-n-Go Buffer at 99 C for 20 minutes Copan 4N6- FLOQSwabs OmniSwabs Puritan swabs Figure 18 Amplification of buccal cells on fresh Copan 4N6FLOQSwabs and lysed with Prep-n-Go Buffer at room temperature AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide 85

86 5 Chapter 5 Experiments and Results Population data Population data SWGDAM guideline 2.7 Overview Population samples used in these studies The distribution of genetic markers in populations should be determined in relevant population groups. (SWGDAM, July 2003) To interpret the significance of a match between genetically typed samples, you must know the population distribution of alleles at each locus in question. If the genotype of the relevant evidence sample is different from the genotype of a suspects reference sample, then the suspect is excluded as the donor of the biological evidence that was tested. An exclusion is independent of the frequency of the two genotypes in the population. If the suspect and evidence samples have the same genotype, then the suspect is included as a possible source of the evidence sample. The probability that another, unrelated individual would also match the evidence sample is estimated by the frequency of that genotype in the relevant population(s). The Identifiler Kit, prior to the addition of the D8S1179 degenerate primer, was used to generate the population data provided in this section. Samples were collected from individuals throughout the United States with no geographical preference. Population Number of samples Samples provided by African-American 357 Kentucky State Police and the Federal Bureau of U.S. Caucasian 349 Investigation U.S. Hispanic 290 Minnesota Bureau of Criminal Apprehension/Memorial Native American 191 Blood Center of Minneapolis In addition to the alleles that were observed and recorded in the Life Technologies databases, other alleles have been published or reported to Life Technologies by other laboratories (see the STRBase at Identifiler Direct Kit allele frequencies Table 5 shows the Identifiler Direct Kit allele frequencies in four populations, listed as percentages. Table 5 Identifiler Direct Kit allele frequencies Allele African- American (n = 357) U.S. Caucasian (n = 349) U.S. Hispanic (n = 290) Native American (n = 191) CSF1PO AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide

87 Chapter 5 Experiments and Results Population data 5 Allele African- American (n = 357) U.S. Caucasian (n = 349) U.S. Hispanic (n = 290) Native American (n = 191) D2S D3S1358 < AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide 87

88 5 Chapter 5 Experiments and Results Population data Allele African- American (n = 357) U.S. Caucasian (n = 349) U.S. Hispanic (n = 290) Native American (n = 191) D5S D7S D8S AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide

89 Chapter 5 Experiments and Results Population data 5 Allele African- American (n = 357) U.S. Caucasian (n = 349) U.S. Hispanic (n = 290) Native American (n = 191) D13S D16S D18S AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide 89

90 5 Chapter 5 Experiments and Results Population data Allele African- American (n = 357) U.S. Caucasian (n = 349) U.S. Hispanic (n = 290) Native American (n = 191) D19S D21S AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide

91 Chapter 5 Experiments and Results Population data 5 Allele African- American (n = 357) U.S. Caucasian (n = 349) U.S. Hispanic (n = 290) Native American (n = 191) FGA AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide 91

92 5 Chapter 5 Experiments and Results Population data Allele African- American (n = 357) U.S. Caucasian (n = 349) U.S. Hispanic (n = 290) Native American (n = 191) TH TPOX AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide

93 Chapter 5 Experiments and Results Population data 5 Allele African- American (n = 357) U.S. Caucasian (n = 349) U.S. Hispanic (n = 290) Native American (n = 191) vwa A minimum allele frequency (0.7% for the African-American database, 0.7% for the U.S. Caucasian database, 0.9% for the U.S. Hispanic database, and 1.3% for the Native American database) is suggested by the National Research Council in forensic calculations. Low-frequency alleles Some alleles of the Identifiler Direct Kit loci occur at a low frequency. For these alleles, a minimum frequency (5 divided by 2n, where n equals the number of individuals in the database) was assigned for the Identifiler Direct Kit African- American, Asian, U.S. Caucasian, and U.S. Hispanic databases, as suggested in the 1996 report of the Committee on DNA Forensic Science (National Research Council, 1996). These databases are summarized in Table 5 on page 86. The minimum reportable genotype frequency at each locus is: for the African-American database; for the U.S. Caucasian database; for the U.S. Hispanic database; and for the Native American database [p2 + p(1 p) θ, where θ = 0.01]. Hence, the minimum combined multilocus genotype frequency at 15 loci is: for the African-American database; for the U.S. Caucasian database; for the U.S. Hispanic database; and 1.23 x for the Native American database. AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide 93

94 5 Chapter 5 Experiments and Results Population data Evaluation of Hardy-Weinberg equilibrium Estimates of expected heterozygosity (HExp) were computed as described by Nei, M. (1973) using the program PopGene Possible divergence from Hardy-Weinberg expectations (HWE) was tested using various methods: by calculating the unbiased estimate of the expected homozygote/heterozygote frequencies (Levene, H., Nei, M. 1978) and using chi-square (HW X 2 p) and likelihood ratio (HW G 2 p) tests (as implemented in the program PopGene 1.32): and with an exact test (HW Exact p), which is a Markov chain method, based on 1000 shuffling experiments, to estimate without bias the exact P-value of the Hardy-Weinberg test with multiple alleles (Guo, S.W. 1992), as implemented in the program GenePop 3.4. An inter-class correlation test analysis (Burrows' composite measure of linkage disequilibria between pairs of loci and X 2 tests for significance [Weir, B. 1990]) was performed separately in each population to detect any correlations between alleles at any of the pair-wise comparisons of the 15 loci, using the program PopGene Observed heterozygosity (H o ), expected heterozygosity, information content, and tests for detecting departures from Hardy-Weinberg equilibrium are shown for each population in Table 6. While a number of the chi-square tests gave seemingly significant p-values (putatively indicating departures from Hardy-Weinberg equilibrium), chi-squared tests are very sensitive to small expected values (as in the case of multiple rare alleles where the expected number of certain genotypes is 1 or fewer, such as with some of these markers), and can greatly inflate the test statistic in this situation (Weir, B. 1990). With the exact test, the number of tests with p-value < 0.05 were 0 in the African American and U.S. Caucasian populations, 1 in the U.S. Hispanic population (D8S1179; p=0.0304) and 2 in the Native Americans (D21S11, p=0.0118; D5S818, p=0.0205). These are no more than would be expected by chance. No more alleles were observed to be in linkage disequilibrium than would be expected by chance alone. The average observed heterozygosity across the 15 STR loci was in the African American population, in the U.S. Caucasian sample population, in the Hispanic sample population, and in the Native Americans. The most heterozygous locus was FGA (mean observed heterozygosity across all populations of 0.875), and the least heterozygous STR locus was TPOX (mean observed heterozygosity across all populations of 0.677). Table 6 Heterozygosity and p-values for Hardy-Weinberg tests of the 15 Identifiler STR loci in four U.S. populations African- American (n = 357) U.S. Caucasian (n = 349) U.S. Hispanic (n = 290) Native American (n = 191) CSF1P0 HW X 2 p HW G 2 p HW Exact p HExp H o HW X 2 p, probability value of X 2 test for Hardy-Weinberg equilibrium; HW G 2 p, probability value of the G-statistic of the Likelihood Ratio test for multinomial proportions; HW Exact p; A Markov chain unbiased exact test to estimate the P-value of the Hardy-Weinberg test with multiple alleles; Hexp, Expected heterozygosity; H o, observed heterozygosity 94 AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide

95 Chapter 5 Experiments and Results Population data 5 African- American (n = 357) U.S. Caucasian (n = 349) U.S. Hispanic (n = 290) Native American (n = 191) D2S1338 HW X 2 p HW G 2 p HW Exact p HExp H o D3S1358 HW X 2 p HW G 2 p HW Exact p HExp H o D5S818 HW X 2 p HW G 2 p HW Exact p HExp H o D7S820 HW X 2 p HW G 2 p HW Exact p HExp H o D8S1179 HW X 2 p HW G 2 p HW Exact p HExp H o AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide 95

96 5 Chapter 5 Experiments and Results Population data African- American (n = 357) U.S. Caucasian (n = 349) U.S. Hispanic (n = 290) Native American (n = 191) D13S317 HW X 2 p HW G 2 p HW Exact p HExp H o D16S539 HW X 2 p HW G 2 p HW Exact p HExp H o D18S51 HW X 2 p HW G 2 p HW Exact p HExp H o D19S433 HW X 2 p HW G 2 p HW Exact p HExp H o D21S11 HW X 2 p HW G 2 p HW Exact p HExp H o FGA HW X 2 p HW G 2 p HW Exact p HExp H o AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide

97 Chapter 5 Experiments and Results Mutation rate 5 African- American (n = 357) U.S. Caucasian (n = 349) U.S. Hispanic (n = 290) Native American (n = 191) TH01 HW X 2 p HW G 2 p HW Exact p HExp H o TPOX HW X 2 p HW G 2 p HW Exact p HExp H o vwa HW X 2 p HW G 2 p HW Exact p HExp H o Concordance studies We compared allele calls between the Identifiler and Identifiler Direct Kits. The genotype data from the 200 analyzed treated paper workflow samples showed 100% concordance between the Identifiler and Identifiler Direct Kits. The genotype data from 84 buccal samples processed using Prep-n-Go Buffer and the Identifiler Direct Kit showed 100% concordance to allele calls generated for purified DNA samples analyzed with the Identifiler kit. Mutation rate Estimation of spontaneous or induced germline mutation at genetic loci can be achieved by comparing the genotypes of offspring to those of their parents. From such comparisons the number of observed mutations are counted directly. In previous studies, genotypes of ten STR loci that were amplified by the AmpFlSTR SGM Plus PCR Amplification Kit were determined for a total of 146 parent-offspring allelic transfers (meioses) at the Forensic Science Service, Birmingham, England. One length-based STR mutation was observed at the D18S11 locus; mutations were not detected at any of the other nine STR loci. The D18S11 mutation was represented by an increase of one 4-nt repeat unit, allele 17 was inherited as allele 18 (single-step mutation). The maternal/paternal source of this mutation could not be distinguished. AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide 97

98 5 Chapter 5 Experiments and Results Probability of identity Additional mutation studies Additional studies (Edwards et al., 1991; Edwards et al., 1992; Weber and Wong, 1993; Hammond et al., 1994; Brinkmann et al., 1995; Chakraborty et al., 1996; Chakraborty et al., 1997; Brinkmann et al., 1998; Momhinweg et al., 1998; Szibor et al., 1998) of direct mutation rate counts produced: Larger sample sizes for some of the Identifiler Direct Kit loci. Methods for modifications of these mutation rates (to infer mutation rates indirectly for those loci where the rates are not large enough to be measured directly and/or to account for those events undetectable as Mendelian errors). Probability of identity Table 7 shows the Probability of Identity (P I ) values of the Identifiler Direct Kit loci individually and combined. Table 7 Probability of Identity values for the Identifiler Direct Kit STR loci Locus African- American U.S. Caucasian U.S. Hispanic Native American CSF1PO D2S D3S D5S D7S D8S D13S D16S D18S D19S D21S FGA TH TPOX vwa Combined The P I value is the probability that two individuals selected at random will have an identical Identifiler Direct Kit genotype (Sensabaugh, 1982). The P I values for the populations described in this section are then approximately 1/ (African- American), 1/ (U.S. Caucasian), 1/ (U.S. Hispanic), and 1/ (Native American). 98 AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide

99 Chapter 5 Experiments and Results Probability of paternity exclusion 5 Probability of paternity exclusion Table 8 shows the Probability of Paternity Exclusion (P E ) values of the Identifiler Direct Kit STR loci individually and combined. Table 8 Probability of Paternity Exclusion values for the Identifiler Direct Kit loci Locus African- American U.S. Caucasian U.S. Hispanic Native American CSF1PO D2S D3S D5S D7S D8S D13S D16S D18S D19S D21S FGA TH TPOX vwa Combined The P E value is the probability, averaged over all possible mother-child pairs, that a random alleged father will be excluded from paternity after DNA typing of the Identifiler Direct Kit STR loci (Chakraborty and Stivers, 1996). AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide 99

100 5 Chapter 5 Experiments and Results Probability of paternity exclusion 100 AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide

101 A Troubleshooting Follow the actions recommended in this appendix to troubleshoot problems that occur during analysis. Table 9 Troubleshooting Observation Possible causes Recommended actions Faint or no signal from both the AmpFlSTR Identifiler Direct Control DNA 9947A and the DNA test samples at all loci Incorrect volume or absence of Identifiler Direct Master Mix or Identifiler Direct Primer Set No activation of AmpliTaq Gold DNA Polymerase Master Mix not vortexed thoroughly before aliquoting Identifiler Direct Primer Set exposed to too much light PCR System malfunction Use of incorrect thermal cycling parameters MicroAmp Base used with tray/ retainer set and tubes in GeneAmp 9700 Insufficient PCR product electrokinetically injected Degraded formamide Sample punch location was not optimal Insufficient lysis of the swab head Repeat amplification. Repeat amplification, making sure to hold reactions initially at 95 C for 11 minutes. Vortex the Master Mix thoroughly. Store the Primer Set protected from light. Refer to the thermal cycler user s manual and check instrument calibration. Check the protocol for correct thermal cycling parameters. Remove MicroAmp Base from tray/retainer set and repeat test. Prepare PCR product as described in Chapter 3, Perform Electrophoresis on page 27. Check the storage of formamide; do not thaw and refreeze multiple times. Try Hi-Di Formamide. For blood samples on treated paper, punch in the center of the blood stain. For buccal samples on treated paper, punch in the center of the buccal transfer or punch in the optimal spot based on past experiences. For buccal samples collected with the Bode Buccal DNA Collector, punch from near the tip of the collector. Ensure swab heads are incubated for 20 minutes in 400 µl Prep-N-Go Buffer. AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide 101

102 A Appendix A Troubleshooting Observation Possible causes Recommended actions More than two alleles present at a locus Some but not all loci visible on electropherogram of DNA Test Samples STR profiles contain many off-scale alleles Presence of exogenous DNA Amplification of stutter product (n-4 nt position) Incomplete 3 A base addition (n-1 nt position) Signal exceeds dynamic range of instrument (off-scale data) Poor spectral separation (bad matrix) Contamination carried over from the disc punching tool Incomplete denaturation of double stranded DNA Disc size used in the amplification reaction was greater than 1.2 mm Insufficient volume of swab lysate added to the reaction Less than 25 µl of PCR reaction volume was used PCR cycle number was too high For blood samples: Too much liquid blood was spotted onto paper substrate Use appropriate techniques to avoid introducing foreign DNA during laboratory handling. See Stutter products on page 73. See Addition of 3 A nucleotide on page 78. Be sure to include the final extension step of 60 C for 10 minutes in the PCR. Ensure cycle number is optimized according to instructions on page 17. Repeat PCR amplification using fewer PCR cycles or use your laboratory s SOP to analyze off-scale data. Follow the steps for creating a spectral file. Confirm that Filter Set G5 modules are installed and used for analysis. Clean the disc punching tool thoroughly. If necessary, include a blank punch step in between the sample punches. Use recommended amount of Hi-Di Formamide and perform heat denaturation step according to the instructions in Chapter 3, Perform Electrophoresis. Repeat amplification using a use 1.2 mm punch size. Repeat amplification using the recommended lysate input volume. Repeat amplification using the recommended PCR reaction volume of 25 µl. Perform sensitivity experiment (page 17) to determine the optimal PCR cycle number based on the sample type. Spot <100 µl of liquid blood per sample area. 102 AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide

103 B Ordering Information Equipment and materials not included Table 10 and Table 11 list required and optional equipment and materials not supplied with the Identifiler Direct Kit. Unless otherwise noted, many of the items are available from major laboratory suppliers (MLS). Table 10 Equipment Equipment Source 3100/3100-Avant Genetic Analyzer Contact your local Life Technologies sales Applied Biosystems 3500/3500xL Genetic Analyzer representative Applied Biosystems 3130/3130xl Genetic Analyzer Applied Biosystems 3730 Genetic Analyzer GeneAmp PCR System 9700 with the Silver 96-Well Block N GeneAmp PCR System 9700 with the Gold-plated Silver 96-Well Block Silver 96-Well Sample Block N Gold-plated Silver 96-Well Sample Block Veriti 96-Well Thermal Cycler ProFlex 96-Well PCR System Tabletop centrifuge with 96-Well Plate Adapters (optional) MLS Harris Manual Punch, 1.2 mm MLS CPA200 Semi-Automated Punch Instrument with a 1.2 mm punch head Contact your local Life CPA300 Fully-Automated Punch Instrument with a 1.2 mm punch head Technologies support representative for information. Bode Buccal DNA Collector This part number is not available for sale in the US. Copan FLOQSwabs Contact your local Life Technologies support representative for information. AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide 103

104 B Appendix B Ordering Information Equipment and materials not included Equipment Source Copan NUCLEIC-CARD system Contact your local Life Technologies support representative for information. This product is not available for sale in the US. 96-well, deep-well plate Table 11 User-supplied materials Item Source AmpFlSTR Identifiler Direct PCR Amplification Kit, 200 reaction AmpFlSTR Identifiler Direct PCR Amplification Kit, 1000 reaction Prep-n-Go Buffer (untreated paper substrate) Prep-n-Go Buffer (buccal swab) Analyzer materials 96-Well Plate Septa Reservoir Septa /3130xl Genetic Analyzer Capillary Array, 36-cm POP-4 Polymer for 3100/3100-Avant Genetic Analyzers /3100-Avant Genetic Analyzer Autosampler Plate Kit, 96-well GeneScan 500 LIZ Size Standard OR GeneScan 600 LIZ Size Standard v OR Running Buffer, Hi-Di Formamide DS-33 Matrix Standard Kit (Dye Set G5) MicroAmp Optical 96-Well Reaction Plate N µL Glass Syringe (array-fill syringe) mL Glass Syringe (polymer-reserve syringe) For a complete list of parts and accessories for the 3100 instrument, refer to Appendix B of the 3100 Genetic Analyzer and 3100-Avant Genetic Analyzer User Reference Guide (Part no ). 3130xl Analyzer materials 96-Well Plate Septa Reservoir Septa /3130xl Genetic Analyzer Capillary Array, 36-cm POP-4 Polymer for 3130/3130xl Genetic Analyzers /3100-Avant Genetic Analyzer Autosampler Plate Kit, 96-well AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide

105 Appendix B Ordering Information Equipment and materials not included B Item Source GeneScan 500 LIZ Size Standard OR GeneScan 600 LIZ Size Standard v OR Running Buffer, DS-33 Matrix Standard Kit (Dye Set G5) MicroAmp Optical 96-Well Reaction Plate N Hi-Di Formamide For a complete list of parts and accessories for the 3130xl instrument, refer to Appendix A of the Applied Biosystems 3130/ 3130xl Genetic Analyzers Maintenance, Troubleshooting, and Reference Guide (Part no ). 3500/3500xL Analyzer materials Anode buffer container (ABC) Cathode buffer container (CBC) POP-4 polymer (960 samples) for 3500/3500xL Genetic Analyzers POP-4 polymer (384 samples) for 3500/3500xL Genetic Analyzers DS-33 Matrix Standard Kit (Dye Set G5) GeneScan 600 LIZ Size Standard v Conditioning reagent Capillary array, 36 cm for 3500 Genetic Analyzers Capillary array, 36 cm for 3500xL Genetic Analyzers well retainer & base set (Standard) 3500/3500xL Genetic Analyzers Tube retainer & base set (Standard) for 3500/3500xL Genetic Analyzers Strip Septa for 3500/3500xL Genetic Analyzers Well Septa for 3500/3500xL Genetic Analyzers Septa Cathode Buffer Container, 3500 series For a complete list of parts and accessories for the 3500/3500xL instrument, refer to the Applied Biosystems 3500/3500xL Genetic Analyzer User Guide (PN ) Analyzer materials 3730 DNA Analyzer Capillary Array, 36-cm GeneScan 500 LIZ Size Standard OR GeneScan 600 LIZ Size Standard v OR Hi-Di Formamide Running Buffer, DS-33 Matrix Standard Kit (Dye Set G5) Well Plate Septa MicroAmp Optical 96-Well Reaction Plate N POP-7 Polymer for the 3730 Genetic Analyzer AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide 105

106 B Appendix B Ordering Information Equipment and materials not included Item Source For a complete list of parts and accessories for the 3730 instrument, refer to Appendix A of the Applied Biosystems 3730/ 3730xl DNA Analyzer Getting Started Guide (Part no ). PCR Amplification MicroAmp 96-Well Tray N MicroAmp Reaction Tube with Cap, 0.2-mL N MicroAmp 8-Tube Strip, 0.2-mL N MicroAmp 8-Cap Strip N MicroAmp 96-Well Tray/Retainer Set MicroAmp 96-Well Base N MicroAmp Clear Adhesive Film MicroAmp Optical Adhesive Film MicroAmp Optical 96-Well Reaction Plate N Other user-supplied materials Hi-Di Formamide, 25-mL Aerosol resistant pipette tips MLS Microcentrifuge tubes MLS Pipettors MLS Tape, labeling MLS Tube, 50-mL Falcon MLS Tube decapper, autoclavable MLS Deionized water, PCR grade MLS Vortex MLS For the Safety Data Sheet (SDS) of any chemical not distributed by Life Technologies, contact the chemical manufacturer. Before handling any chemicals, refer to the SDS provided by the manufacturer, and observe all relevant precautions. 106 AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide

107 C Plate Layouts Example PCR plate layout The following layout is recommended for use with the sensitivity experiment on page 17. Create 3 identical plates for amplification at 3 different cycle numbers. Example electrophoresis plate layout The following layout is recommended for use with the sensitivity experiment on page 17. AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide 107

108 C Appendix C Plate Layouts Example electrophoresis plate layout 108 AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide

109 D PCR Work Areas Work area setup and lab design PCR setup work area Amplified DNA work area Work area setup and lab design Many resources are available for the appropriate design of a PCR laboratory. If you are using the AmpFlSTR Identifiler Direct PCR Amplification Kit for: Forensic DNA testing, refer to Forensic Laboratories: Handbook for Facility Planning, Design, Construction and Moving, National Institute of Justice, 1998 Parentage DNA testing, refer to the Guidance for Standards for Parentage Relationship Testing Laboratories, American Association of Blood Banks, 7th edition, 2004 The sensitivity of the Identifiler Direct Kit (and other PCR-based tests) enables amplification of minute quantities of DNA, necessitating precautions to avoid contamination of samples yet to be amplified (Kwok and Higuchi, 1989). Also take care while handling and processing samples to prevent contamination by human DNA. Wear gloves at all times and change them frequently. Close sample tubes when not in use. Limit aerosol dispersal by handling sample tubes and reagents carefully. Note: We do not intend these references for laboratory design to constitute all precautions and care necessary for using PCR technology. PCR setup work area IMPORTANT! These items should never leave the PCR Setup Work Area. Calculator Gloves, disposable Marker pen, permanent Microcentrifuge Microcentrifuge tubes, 1.5-mL, or 2.0-mL, or other appropriate clean tube (for Master Mix preparation) Microcentrifuge tube rack Pipette tips, sterile, disposable hydrophobic filter-plugged Pipettors AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide 109

110 D Appendix D PCR Work Areas Amplified DNA work area Tube decapper, autoclavable Vortex Amplified DNA work area IMPORTANT! Place the thermal cyclers in the Amplified DNA Work Area. You can use the following systems: GeneAmp PCR System 9700 with the Silver 96-Well Block GeneAmp PCR System 9700 with the Gold-plated Silver 96-Well Block IMPORTANT! The Identifiler Direct Kit is not validated for use with the GeneAmp PCR System 9700 with the Aluminium 96-Well Block. Use of this thermal cycling platform may adversely affect performance of the Identifiler Direct Kit. Veriti 96-Well Thermal Cycler 110 AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide

111 E Safety WARNING! GENERAL SAFETY. Using this product in a manner not specified in the user documentation may result in personal injury or damage to the instrument or device. Ensure that anyone using this product has received instructions in general safety practices for laboratories and the safety information provided in this document. Before using an instrument or device, read and understand the safety information provided in the user documentation provided by the manufacturer of the instrument or device. Before handling chemicals, read and understand all applicable Safety Data Sheets (SDSs) and use appropriate personal protective equipment (gloves, gowns, eye protection, etc). To obtain SDSs, see the Documentation and Support section in this document. AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide 111

112 E Appendix E Safety Chemical safety Chemical safety WARNING! GENERAL CHEMICAL HANDLING. To minimize hazards, ensure laboratory personnel read and practice the general safety guidelines for chemical usage, storage, and waste provided below, and consult the relevant SDS for specific precautions and instructions: Read and understand the Safety Data Sheets (SDSs) provided by the chemical manufacturer before you store, handle, or work with any chemicals or hazardous materials. To obtain SDSs, see the Documentation and Support section in this document. Minimize contact with chemicals. Wear appropriate personal protective equipment when handling chemicals (for example, safety glasses, gloves, or protective clothing). Minimize the inhalation of chemicals. Do not leave chemical containers open. Use only with adequate ventilation (for example, fume hood). Check regularly for chemical leaks or spills. If a leak or spill occurs, follow the manufacturer's cleanup procedures as recommended in the SDS. Handle chemical wastes in a fume hood. Ensure use of primary and secondary waste containers. (A primary waste container holds the immediate waste. A secondary container contains spills or leaks from the primary container. Both containers must be compatible with the waste material and meet federal, state, and local requirements for container storage.) After emptying a waste container, seal it with the cap provided. Characterize (by analysis if necessary) the waste generated by the particular applications, reagents, and substrates used in your laboratory. Ensure that the waste is stored, transferred, transported, and disposed of according to all local, state/provincial, and/or national regulations. IMPORTANT! Radioactive or biohazardous materials may require special handling, and disposal limitations may apply. Specific chemical handling CAS Chemical Phrase Sodium Azide Sodium azide may react with lead and copper plumbing to form highly explosive metal azides. Biological hazard safety WARNING! Potential Biohazard. Depending on the samples used on this instrument, the surface may be considered a biohazard. Use appropriate decontamination methods when working with biohazards. 112 AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide

113 Appendix E Safety Biological hazard safety E WARNING! BIOHAZARD. Biological samples such as tissues, body fluids, infectious agents, and blood of humans and other animals have the potential to transmit infectious diseases. Follow all applicable local, state/provincial, and/or national regulations. Wear appropriate protective equipment, which includes but is not limited to: protective eyewear, face shield, clothing/lab coat, and gloves. All work should be conducted in properly equipped facilities using the appropriate safety equipment (for example, physical containment devices). Individuals should be trained according to applicable regulatory and company/ institution requirements before working with potentially infectious materials. Read and follow the applicable guidelines and/or regulatory requirements in the following: In the U.S.: U.S. Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories found at: Occupational Safety and Health Standards, Bloodborne Pathogens (29 CFR ), found at: 29cfr1910a_01.html Your company s/institution s Biosafety Program protocols for working with/ handling potentially infectious materials. Additional information about biohazard guidelines is available at: In the EU: Check local guidelines and legislation on biohazard and biosafety precaution and refer to the best practices published in the World Health Organization (WHO) Laboratory Biosafety Manual, third edition, found at: csr/resources/publications/biosafety/who_cds_csr_lyo_2004_11/en/ AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide 113

114 E Appendix E Safety Biological hazard safety 114 AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide

115 Bibliography Akane, A., Matsubara, K., Nakamura, H., Takahashi, S., and Kimura, K Identification of the heme compound copurified with deoxyribonucleic acid (DNA) from bloodstains, a major inhibitor of polymerase chain reaction (PCR) amplification. J. Forensic Sci. 39: Bonferroni, C.E Teoria statistica delle classi e calcolo Belle probabilita. Publicazioni del R Istituto Superiore di Scienze Economiche e Commerciali di Firenze 8:3 62. Barber, M.D. and Parkin, B.H Sequence analysis and allelic designation of the two short tandem repeat loci D18S51 and D8S1179. Intl. J. Legal Med. 109: Baron, H., Fung, S., Aydin, A., Bahrig, S., Luft, F.C., Schuster, H Oligonucleotide ligation assay (OLA) for the diagnosis of familial hypercholesterolemia. Nat. Biotechnol. 14: Begovich A.B., McClure G.R., Suraj V.C., Helmuth R.C., Fildes N., Bugawan T.L., Erlich H.A., Klitz W Polymorphism, recombination, and linkage disequilibrium within the HLA class II region. J. Immunol. 148: Bender, K., Farfan, M.J., Schneider, P.M Preparation of degraded human DNA under controlled conditions. Forensic Sci. Int. 139: Brinkman, B., Klintschar, M., Neuhuber, F., Huhne, J. and Rolf, B Mutation rate in human microsatellites: Influence of the structure and length of the tandem repeat. Am. J. Hum. Genet. 62: Brinkman, B., Moller, A. and Wiegand, P Structure of new mutations in 2 STR systems. Intl. J. Legal Med. 107: Butler, J.M Forensic DNA Typing. Burlington, MA:Elsevier Academic Press. Butler, J.M., Shen, Y., McCord, B.R The development of reduced size STR amplicons as tools for analysis of degraded DNA. J. Forensic Sci. 48: Chakraborty, R. Kimmel, M., Stivers, D., Davison, L., and Deka, R Relative mutation rates at di-, tri-, and tetranucleotide microsatellite loci. Proc. Natl. Acad. Sci. USA 94: Chakraborty, R., Stivers, D., and Zhong, Y Estimation of mutation rates from parentage exclusion data: applications to STR and VNTR loci. Mutat. Res. 354: Chakraborty, R. and Stivers, D.N Paternity exclusion by DNA markers: effects of paternal mutations. J. Forensic Sci. 41: Chung, D.T., Drabek, J., Opel, K.L., Butler, J.M. and McCord, B.R A study of the effects of degradation and template concentration on the amplification efficiency of the Miniplex primer sets. J. Forensic Sci. 49: Clark J.M Novel non-templated nucleotide addition reactions catalyzed by procaryotic and eucaryotic DNA polymerases. Nucleic Acids Res. 16: AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide 115

116 Bibliography Coble, M.D. and Butler, J.M Characterization of new ministr loci to aid analysis of degraded DNA. J. Forensic Sci. 50: DeFranchis, R., Cross, N.C.P., Foulkes, N.S., and Cox, T.M A potent inhibitor of Taq DNA polymerase copurifies with human genomic DNA. Nucleic Acids Res. 16: DNA Advisory Board, Federal Bureau of Investigation, U.S. Department of Justice Quality assurance standards for forensic DNA testing laboratories. Drabek, J., Chung, D.T., Butler, J.M., McCord, B.R Concordance study between Miniplex assays and a commercial STR typing kit. J. Forensic Sci. 49: Edwards, A., Civitello, A., Hammond, H., and Caskey, C DNA typing and genetic mapping with trimeric and tetrameric tandem repeats. Am. J. Hum. Genet. 49: Edwards, A., Hammond, H.A., Lin, J., Caskey, C.T., and Chakraborty, R Genetic variation at five trimeric and tetrameric tandem repeat loci in four human population groups. Genomics 12: Frank, W., Llewellyn, B., Fish, P., et al Validation of the AmpFlSTR Profiler Plus PCR Amplification Kit for use in forensic casework. J. Forensic Sci. 46: Glock, B., Dauber, E.M., Schwartz, D.W., Mayr W.R Additional variability at the D12S391 STR locus in an Austrian population sample: sequencing data and allele distribution. Forensic Sci. Int. 90: Grossman, P.D., Bloch, W., Brinson, E., Chang, C.C., Eggerding, F.A., Fung, S., Iovannisci, D.M., Woo, S., Winn-Deen, E.S High-density multiplex detection of nucleic acid sequences: oligonucleotide ligation assay and sequence-coded separation. Nucleic Acids Res. 22: Grubwieser, P. Muhlmann, R., Berger, B., Niederstatter, H., Palvic, M., Parson, W A new mini-str-multiplex displaying reduced amplicon lengths for the analysis of degraded DNA. Int. J. Legal Med. 120: Guo, S.W. and Thompson, E.A Performing the exact test of Hardy-Weinberg proportion for multiple alleles. Biometrics 48: Guthmiller, J.M., Vargas, K.G., Srikantha, R., Schomberg, L.L., Weistroffer, P.L., McCray, P.B. and Tack, B.F Susceptibilities of oral bacteria and yeast to mammalian cathelicidins. Antimicrob. Agents Chemother. 45: Hammond, H., Jin, L., Zhong, Y., Caskey, C., and Chakraborty, R Evaluation of 13 short tandem repeat loci for use in personal identification applications. Am J. Hum. Genet. 55: Holt, C., Stauffer, C., Wallin, J., et al Practical applications of genotypic Surveys for forensic STR testing. Forensic Sci. Int. 112: Kalinowski, S.T HW-QuickCheck: an easy-to-use computer program for checking genotypes for agreement with Hardy-Weinberg expectations. Molecular Ecology Notes 6: Kimpton, C., Walton, A., and Gill, P A further tetranucleotide repeat polymorphism in the vwf gene. Hum. Mol. Genet. 1:287. Kong, X., Murphy, K., Raj, T., He, C., White, P.S., Matise, T.C A combined linkage-physical map of the human genome. Am. J. Hum. Genet. 75: AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide

117 Bibliography Kwok, S., and Higuchi, R Avoiding false positives with PCR. Nature 339: Lareu, M.V., Pestoni, M.C., Barros, F., Salas, A., Carracedo, A Sequence variation of a hypervariable short tandem repeat at the D12S391 locus. Gene 182: Lazaruk, K., Walsh, P.S., Oaks, F., Gilbert, D., Rosenblum, B.B., Menchen, S., Scheibler, D., Wenz, H.M., Holt, C., Wallin, J Genotyping of forensic short tandem repeat (STR) systems based on sizing precision in a capillary electrophoresis instrument. Electrophoresis 19: Levene, H On a matching problem in genetics. Ann. Math. Stat. 20: Li, H. Schmidt, L., Wei, M-H., Hustad, T. Leman, M.I., Zbar, B. and Tory, K Three tetranucleotide polymorphisms for loci:d3s1352; D3S1358; D3S1359. Hum. Mol. Genet. 2:1327. Magnuson, V.L., Ally, D.S., Nylund, S.J., Karanjawala, Z.E., Rayman, J.B., Knapp, J.I., Lowe, A.L., Ghosh, S., Collins, F.S Substrate nucleotide-determined nontemplated addition of adenine by Taq DNA polymerase: implications for PCR-based genotyping and cloning. Biotechniques 21: Mansfield, E.S., Robertson, J.M., Vainer, M., Isenberg, A.R., Frazier, R.R., Ferguson, K., Chow, S., Harris, D.W., Barker, D.L., Gill, P.D., Budowle, B., McCord, B.R Analysis of multiplexed short tandem repeat (STR) systems using capillary array electrophoresis. Electrophoresis 19: Mills, K.A., Even, D., and Murrau, J.C Tetranucleotide repeat polymorphism at the human alpha fibrinogen locus (FGA). Hum. Mol. Genet. 1:779. Momhinweg, E., Luckenbach, C., Fimmers, R., and Ritter, H D3S1358: sequence analysis and gene frequency in a German population. Forensic Sci. Int. 95: Moretti, T., Baumstark, A., Defenbaugh, D., Keys, K., Smerick, J., and Budowle, B Validation of short tandem repeats (STRs) for forensic usage: Performance testing of fluorescent multiplex STR systems and analysis of authentic and simulated forensic samples. J. Forensic Sci. 46(3): Mulero, J.J., Chang, C.W., and Hennessy, L.K Characterization of N+3 stutter product in the trinucleotide repeat locus DYS392. J. Forensic Sci. 51: Nakahori, Y., Takenaka, O., and Nakagome, Y A human X-Y homologous region encodes amelogenin. Genomics 9: Nei, M Analysis of gene diversity in subdivided populations. Proc. Natl. Acad. Sci. USA 70: Nei, M Estimation of average heterozygosity and genetic distance from a small number of individuals. Genetics 89: Revised Validation Guidelines-Scientific Working Group on DNA Analysis Methods (SWGDAM). Forensic Science Communications (July 2004) Volume 6 (3). Available at Sensabaugh, G.F Biochemical markers of individuality. In: Saferstein, R., ed. Forensic Science Handbook. Prentice-Hall, Inc., New York, pp Sharma, V. and Litt, M Tetranucleotide repeat polymorphism at the D21S11 locus. Hum Mol. Genet. 1:67. Shin, C.H., Jang, P., Hong, K.M,, Paik, M.K Allele frequencies of 10 STR loci in Koreans. Forensic Sci. Int. 140: AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide 117

118 Bibliography Smith, R.N Accurate size comparison of short tandem repeat alleles amplified by PCR. Biotechniques 18: Sparkes, R., Kimpton, C., Watson, S., Oldroyd, N., Clayton, T., Barnett, L., Arnold, J., Thompson, C., Hale, R., Chapman, J., Urquhart, A., and Gill, P. 1996a. The validation of a 7-locus multiplex STR test for use in forensic casework. (I). Mixtures, ageing, degradation and species studies. Int. J. Legal Med. 109: Sparkes, R., Kimpton, C., Gilbard, S., Carne, P., Andersen, J., Oldroyd, N., Thomas, D., Urquhart, A., and Gill, P. 1996b. The validation of a 7-locus multiplex STR test for use in forensic casework. (II), Artifacts, casework studies and success rates. Int. J. Legal Med. 109: Straub, R.E., Speer, M.C., Luo, Y., Rojas, K., Overhauser, J., Ott, J., and Gilliam, T.C A microsatellite genetic linkage map of human chromosome 18. Genomics 15: Suido, H., Nakamura, M., Mashimo, P.A., Zambon, J.J., and Genco, R.J Arylaminopeptidase activities of the oral bacteria. J. Dent. Res. 65: Waiyawuth, W., Zhang, L., Rittner, C., Schneider, P.M Genetic analysis of the short tandem repeat system D12S391 in the German and three Asian populations. Forensic Sci. Int. 94: Wallin, J.M., Buoncristiani, M.R., Lazaruk, K.D., Fildes, N., Holt, C.L., Walsh, P.S SWGDAM validation of the AmpFlSTR blue PCR amplification kit for forensic casework analysis. J. Forensic Sci. 43: Wallin, J.M., Holt, C.L., Lazaruk, K.D., Nguyen, T.H., Walsh, P.S Constructing universal multiplex PCR systems for comparative genotyping. J. Forensic Sci. 47: Walsh, P.S., Fildes, N.J., Reynolds, R Sequence analysis and characterization of stutter products at the tetranucleotide repeat locus vwa. Nucleic Acids Res. 24: Weber, J. and Wong, C Mutation of human short tandem repeats. Hum. Mol. Genet. 2: Weir, B Genetic Data Analysis. Sinauer Associates Sunderland, MA Wiegand, P. and Kleiber, M Less is more length reduction of STR amplicons using redesigned primers. Int. J. Legal Med. 114: AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide

119 Documentation and Support Related documentation Document title Part number 3100/3100-Avant Data Collection v2.0 User Guide /3100-Avant Genetic Analyzers Using Data Collection Software v2.0 User Bulletin Genetic Analyzer User Manual (Data Collection v1.1) /3100-Avant Genetic Analyzers Protocols for Processing AmpFlSTR PCR Amplification Kit PCR Products User Bulletin Veriti 96-Well Thermal Cycler AmpFlSTR Kit Validation User Bulletin ProFlex PCR System Kit Validation User Bulletin Applied Biosystems 3130/3100xl Genetic Analyzers Using Data Collection Software v3.0 User Bulletin Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide Applied Biosystems 3130/3130xl Genetic Analyzers Maintenance, Troubleshooting, and Reference Guide Applied Biosystems 3130/3130xl Genetic Analyzers Quick Reference Card Applied Biosystems 3130/3130xl Genetic Analyzers AB Navigator Software Administrator Guide Applied Biosystems 3130/3100xl DNA Analyzers User Guide Applied Biosystems 3500/3500xL Genetic Analyzer Quick Reference Card Applied Biosystems 3500/3500xL Genetic Analyzer User Guide, Data Collection v Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin: Solutions to issues related to software, data, hardware, and consumables Note: Additional user bulletins may be available at Applied Biosystems 3730/3730xl Genetic Analyzer Getting Started Guide GeneAmp PCR System 9700 Base Module User s Manual N Quantifiler Kits: Quantifiler Human DNA Quantification Kit and Quantifiler Y Human Male DNA Quantification Kit User s Manual AmpFlSTR Identifiler PCR Amplification Kit User s Manual GeneMapper ID Software Version 3.1 Human Identification Analysis User Guide GeneMapper ID Software Versions 3.1 and 3.2 Human Identification Analysis Tutorial Installation Procedures and New Features for GeneMapper ID Software v3.2 User Bulletin GeneMapper ID-X Software Version 1.0 Getting Started Guide GeneMapper ID-X Software Version 1.0 Quick Reference Guide GeneMapper ID-X Software Version 1.0 Reference Guide GeneMapper ID-X Software Version 1.1 (Mixture Analysis) Getting Started Guide AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide 119

120 Documentation and Support Obtain support Document title Part number GeneMapper ID-X Software Version 1.1 (Mixture Analysis) Quick Reference Guide GeneMapper ID-X Software Version 1.2 Reference Guide GeneMapper ID-X Software Version 1.2 Quick Reference Guide Portable document format (PDF) versions of this guide and the documents listed above are available at Note: To open the user documentation available from the our web site, use the Adobe Acrobat Reader software available from Obtain support For HID support: In North America Send an to or call option 1. Outside North America Contact your local support office. For the latest services and support information for all locations, go to: At the website, you can: Access worldwide telephone and fax numbers to contact Technical Support and Sales facilities Search through frequently asked questions (FAQs) Submit a question directly to Technical Support Search for user documents, SDSs, vector maps and sequences, application notes, formulations, handbooks, certificates of analysis, citations, and other product support documents Obtain information about customer training Download software updates and patches Limited Product Warranty Life Technologies Corporation and/or its affiliate(s) warrant their products as set forth in the Life Technologies' General Terms and Conditions of Sale found on Life Technologies website at If you have any questions, please contact Life Technologies at AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide

121 Index Symbols.fsa sample files 36, 49.hid sample file 48 +A nucleotide addition defined 78 efficiency of 78 Numerics 3100/3100-Avant instruments /3500xL instruments instrument 31 A accuracy and reproducibility 64 alleles low frequency 93 off-ladder 65 allelic ladder figure 11 requirements for accurate genotyping 28 volume per reaction 30, 32, 34 analysis method 40, 53 analysis method, create 40 analyze a project 46, 60 artifacts in data 79 B bins import 49 bins, import 36, 49 biohazard safety 112 blood samples 9, 17, 19 Bode Buccal DNA Collector 9, 17, 21 buccal samples 9, 17, 19, 21 buccal swabs 23 C characterization of loci, validation 79 AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide chemical safety 112 concordance studies 97 contents of kit 14 control DNA, about 15 Copan FLOQSwabs 85 treated cards 9 D data collection software 13 data, accuracy, precision, and reproducibility of 64 data, artifacts 79 data, for different populations 86 developmental validation 64 DNA effect of quantity, figure 83 negative control sample preparation 19, 21, 24 positive control sample preparation 19, 21, 24 sensitivity 81 your sample preparation 19, 21, 24 documentation, related 119 E electropherogram causes of extra peaks 73 extra peaks 73 species specificity 80, 84, 85 electrophoresis Data Collection Software 29, 31 data collection software 33 prepare samples 30, 31, 33 references 29, 31, 33 run module 29, 31, 33 set up of 3100/3100-Avant instruments 29 set up of 3500/3500xL instruments 31 set up of 3730 instrument 31, 33 emission spectra 14 equipment, not included with kit 103 extra peaks, causes

122 Index F five-dye fluorescent system 10 fluorescent dyes 13 FSA sample files 36, 49 FTA cards 9, 17, 19 G GeneMapper ID Software analyze a project 46 analyze files 46 data analysis 36 edit a project 47 examine a project 47 import panels and bins 36 size standard, create 45 GeneMapper ID-X Software analyze a project 60 analyze files 60 data analysis 49 marker stutter 49 panels, bins, and stutter, import 49 size standard, create 58 GeneScan size standard about 15 dye label 13 volume per reaction 30, 31, 33 GS GS H Hi-Di formamide, volume per reaction 30, 31, 33 I import bins 36 panels 36 panels, bins, stutter 49 size standard 45, 58 Instrument and software compatibility 13 Instrument and special software compatibility 13 K kit contents 14 description 9 fluorescent dyes 13 L instruments for use with 9 loci amplified 10 master mix 14 primers 10, 14 purpose 9 reagents 14 storage 14 thermal cyclers for use with 110 Limited Product Warranty 120 LIZ size standard about 15 volume per reaction 30, 31, 33 loci characterization 79 chromosomal location 10 combined genotype frequency 93 dye label 10 genotype frequency in population 93 mapping 80 lysate, prepare 23 M marker stutter, import 49 master mix, volume per reaction 19, 21, 23 materials and equipment 14 materials, not included with kit 103 multicomponent analysis 13 mutation studies 98 mutation, STR 97 N normalization 56 O off-ladder alleles 65 operating systems 29, 31, 33 P panels, import 36, 49 PCR optimize cycle number 17 perform 26 setup AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide

123 Index PCR work areas 103, 109 percent stutter off-scale peaks 74 relation to allele length 73 precision and size windows 65 precision, sizing 65 Prep-n-Go Buffer 9, 17, 21 primers about 10 Amelogenin 79 volume per reaction 19, 21, 23 probability of identity definition 98 values 98 project examination and editing 61 punches, size 19, 21 R reaction mix, for PCR 20, 22, 24 reactions, prepare for PCR 19, 21, 23 reagents not included with kit 103 run module, electrophoresis 29, 31, 33 S safety biohazard 112 chemical 112 sample discs, size 19, 21 sample files,.fsa 36, 49 size deviation, sample alleles and ladder alleles 64 size standard create 45, 58 GS 50 and GS size standard, create 45, 58 sizing precision 65 species specificity 80 split peaks, +A nucleotide addition 78 standards for samples 15 STRBase 86 stutter products 73 substrates swab 23 treated paper 19 untreated paper 21 support, obtaining 120 swab PCR 26 T prepare reactions 23 sample preparation guidelines 23 samples 9 types 9 validation 85 technical support 120 Terms and Conditions 120 thermal cyclers for use with kit 110 programming 26 training, information on 120 treated paper 19 PCR 26 prepare reactions 19 sample preparation guidelines 19 U untreated paper 21 PCR 26 prepare reactions 21 sample preparation guidelines 21 V validation characterization of loci 79 developmental 64 effect of DNA quantity 82 experiments to evaluate 63 importance of 63 mutation rate 97 population data 86 probability of identity 98 probability of paternity exclusion 99 sensitivity 81 size deviation, sample and ladder alleles 64 species specificity 80 swabs 85 W warranty 120 work area amplified DNA 107, 110 PCR setup 109 setup and lab design 109 workflow overview 12 AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide 123

124 Index 124 AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide

125

126 Headquarters 5791 Van Allen Way Carlsbad, CA USA Phone Toll Free in USA For support visit February 2015